Method of treating non-small-cell lung cancer using pharmaceutical formulation containing thienotriazolodiazepine compounds

ABSTRACT

A method of treating non-small cell lung cancer in a mammal by administering to a patient a pharmaceutically acceptable amount of a compound being a thienotriazolodiazepine compound of the Formula (1) wherein R 1  is alkyl having a carbon number of 1-4, R 2  is a hydrogen atom; a halogen atom; or alkyl having a carbon number of 1-4 optionally substituted by a halogen atom or a hydroxyl group, R 3  is a halogen atom; phenyl optionally substituted by a halogen atom, alkyl having a carbon number of 1-4, alkoxy having a carbon number of 1-4 or cyano; —NR 5 —(CH 2 ) m —R 6  wherein R 5  is a hydrogen atom or alkyl having a carbon number of 1-4, m is an integer of 0-4, and R 6  is phenyl or pyridyl optionally substituted by a halogen atom; or —NR 7 —CO—(CH 2 ) n —R 8  wherein R 7  is a hydrogen atom or alkyl having a carbon number of 1-4, n is an integer of 0-2, and R 8  is phenyl or pyridyl optionally substituted by a halogen atom, and R 4  is —(CH 2 ) a —CO—NH—R 9  wherein a is an integer of 1-4, and R 9  is alkyl having a carbon number of 1-4; hydroxyalkyl having a carbon number of 1-4; alkoxy having a carbon number of 1-4; or phenyl or pyridyl optionally substituted by alkyl having a carbon number of 1-4, alkoxy having a carbon number of 1-4, amino or a hydroxyl group or —(CH 2 ) b —COOR 10  wherein b is an integer of 1-4, and R 10  is alkyl having a carbon number of 1-4, or a pharmaceutically acceptable salt thereof or a hydrate or solvate thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/909,599 filed Nov. 27, 2013, 62/012,042 filed Jun. 13, 2014 and62/080,779 filed Nov. 17, 2014, each of which is incorporated in itsentirety.

FIELD OF INVENTION

The present disclosure describes a method of treating non-small celllung cancer using thienotriazolodiazepine compounds which have improvedsolubility and bioavailability which may be provided in the form ofsolid dispersions.

BACKGROUND OF THE INVENTION

The compound of Formula (1), described herein below, has been shown toinhibit the binding of acetylated histone H4 to the tandem bromodomain(BRD)-containing family of transcriptional regulators known as the BET(bromodomains and extraterminal) proteins, which include BRD2, BRD3, andBRD4. See U.S. Patent Application Publication No. 2010/0286127 A1, whichis incorporated herein by reference in its entirety. The BET proteinshave emerged as major epigenetic regulators of proliferation anddifferentiation and also have been associated with predisposition todyslipidemia or improper regulation of adipogenesis, elevatedinflammatory profile and risk for cardiovascular disease and type 2diabetes, and increased susceptibility to autoimmine diseases such asrheumatoid arthritis and systemic lupus erythematosus as reported byDenis, G. V. “Bromodomain coactivators in cancer, obesity, type 2diabetes, and inflammation,” Discov Med 2010; 10:489-499, which isincorporated herein by reference in its entirety. Accordingly, thecompound of formula (II) may be useful for treatment of various cancers,cardiovascular disease, type 2 diabetes, and autoimmune disorders suchas rheumatoid arthritis and systemic lupus erythematosus.

The compound of Formula (1), described herein below, has been shown toinhibit the binding of acetylated histone H4 to the tandem bromodomain(BRD)-containing family of transcriptional regulators known as the BET(bromodomains and extraterminal) proteins, which include BRD2, BRD3, andBRD4. See U.S. Patent Application Publication No. 2010/0286127 A1, whichis incorporated herein by reference in its entirety. The BET proteinshave emerged as major epigenetic regulators of proliferation anddifferentiation and also have been associated with predisposition todyslipidemia or improper regulation of adipogenesis, elevatedinflammatory profile and risk for cardiovascular disease and type 2diabetes, and increased susceptibility to autoimmine diseases such asrheumatoid arthritis and systemic lupus erythematosus as reported byDenis, G. V. “Bromodomain coactivators in cancer, obesity, type 2diabetes, and inflammation,” Discov Med 2010; 10:489-499, which isincorporated herein by reference in its entirety. Accordingly, thecompound of formula (II) may be useful for treatment of various cancers,cardiovascular disease, type 2 diabetes, and autoimmune disorders suchas rheumatoid arthritis and systemic lupus erythematosus.

The thienotriazolodiazepine compound of Formula (1), described hereinbelow, presents highly specific difficulties in relation toadministration generally and the preparation of galenic compositions inparticular, including the particular problems of drug bioavailabilityand variability in inter- and intra-patient dose response, necessitatingdevelopment of a non-conventional dosage form with respect to thepractically water-insoluble properties of the thienotriazolodiazepine.

Previously, it had been found that thienotriazolodiazepine compound ofFormula (1) could be formulated with the carrier ethyl acrylate-methylmethacrylate-trimethylammonioethyl methacrylate chloride copolymer(Eudragit RS, manufactured by Rohm) to provide an oral formulation thatpreferentially released the pharmaceutical ingredient in the lowerintestine for treatment of inflammatory bowel diseases such asulcerative colitis and Crohn's disease as reported in U.S. PatentApplication Publication No. 20090012064 A1, which is incorporated hereinby reference in its entirety. Through various experiments includinganimal tests, it was found that for inflammatory bowel diseases, thethienotriazolodiazepine compound of Formula (1) release in a lesion anda direct action thereof on the inflammatory lesion were more importantthan the absorption of thienotriazolodiazepine compound of Formula (1)into circulation from the gastrointestinal tract. However, for manyother disease conditions high absorption of thienotriazolodiazepinecompound of Formula (1) into the circulation from gastrointestinal tractis required. Accordingly, a need exists for formulations ofthienotriazolodiazepine compound of Formula (1) that can provide highabsorption of thienotriazolodiazepine compound of Formula (1) into thecirculation from gastrointestinal tract.

Various inhibitors targeting the activity of BET proteins have beenrecently developed and have shown potent anti-proliferative effects inseveral tumors including non-small cell lung cancer (“NSCLC”). Thefusion of the echinoderm microtubule-associated protein-like 4 (EML4)and the anaplastic lymphoma kinase (ALK) genes results in the chimericoncogene EML4-ALK identified a distinct entity of NSCLC exhibiting ahigh rate of therapeutic activity to ALK inhibitors, but most patientsinvariably acquire resistance within few months. Here, we report thepreclinical findings obtained with OTX015, a novel oral pan-BET-BRDinhibitor in a panel of NSCLC cell lines, some of them bearing thefusion protein EML4-ALK.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present disclosure provides for methods oftreating NSCLC using the compositions described herein.

In one embodiment, the present disclosure provides for a method oftreating non-small cell lung cancer in a mammal comprising:administering to a patient in need a pharmaceutically acceptable amountof a composition comprising a solid dispersion according to thecompositions described in sections III, IV, V, VI and VII.

In some embodiments the method further comprises administering to thepatient an mTOR inhibitor. In one such embodiment the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus,ridaforolimus and everolimus. In one such embodiment the mTOR inhibitoris everolimus. The thienotriazolodiazepine compound and the mTORinhibitor can be administered simultaneously or sequentially. In someembodiments such combination of thienotriazolodiazepine compound andmTOR inhibitor produces a synergistic effect.

In some embodiments the method further comprises administering to thepatient an ALK inhibitor. In one such embodiment the ALK inhibitor isselected from the group consisting of ceritinib and crizotinib. In onesuch embodiment the ALK inhibitor is crizotinib. Thethienotriazolodiazepine compound and the ALK inhibitor can beadministered simultaneously or sequentially. In some embodiments suchcombination of thienotrizolodiazepine compound and ALK inhibitorproduces a synergistic effect.

In one embodiment, the non-small cell lung cancer is EML4-ALk positive.In one such embodiment, the non-small cell lung cancer exhibited downregulation of N-MYC mRNA levels after treatment. In another suchembodiment, the non-small cell lung cancer expressed BRD4/3/2, c-MYC,BCL-2, p21 and CyclinD1. In still another embodiment, the treatmentinduced a transient up-regulation of STATS with a subsequentdown-regulation after 24 hour and up to 72 hour exposure.

In still another embodiment, the non-small cell lung cancer is EML4-ALknegative. In one such embodiment, the non-small cell lung cancerexpressed BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1. In another suchembodiment, the treatment induced a transient up-regulation of STATSwith a subsequent down-regulation after 24 hour and up to 72 hourexposure.

In one embodiment, the present disclosure provides for methods oftreating NSCLC using a thienotriazolodiazepine compound of the Formula(1)

whereinR¹ is alkyl having a carbon number of 1-4, R² is a hydrogen atom; ahalogen atom; or alkyl having a carbon number of 1-4 optionallysubstituted by a halogen atom or a hydroxyl group, R³ is a halogen atom;phenyl optionally substituted by a halogen atom, alkyl having a carbonnumber of 1-4, alkoxy having a carbon number of 1-4 or cyano;—NR⁵—(CH₂)_(m)—R⁶ wherein R⁵ is a hydrogen atom or alkyl having a carbonnumber of 1-4, m is an integer of 0-4, and R⁶ is phenyl or pyridyloptionally substituted by a halogen atom; or —NR⁷—CO—(CH₂)_(n)—R⁸wherein R⁷ is a hydrogen atom or alkyl having a carbon number of 1-4, nis an integer of 0-2, and R⁸ is phenyl or pyridyl optionally substitutedby a halogen atom, and R⁴ is —(CH₂)_(a)—CO—NH—R⁹ wherein a is an integerof 1-4, and R⁹ is alkyl having a carbon number of 1-4; hydroxyalkylhaving a carbon number of 1-4; alkoxy having a carbon number of 1-4; orphenyl or pyridyl optionally substituted by alkyl having a carbon numberof 1-4, alkoxy having a carbon number of 1-4, amino or a hydroxyl groupor —(CH₂)_(b)—COOR¹⁰ wherein b is an integer of 1-4, and R¹⁰ is alkylhaving a carbon number of 1-4, including any salts, isomers,enantiomers, racemates, hydrates, solvates, metabolites, and polymorphsthereof.

In some embodiments, Formula (1) is selected from Formula (1A):

wherein X is a halogen, R¹ is C₁-C₄ alkyl, R² is C₁-C₄ alkyl, a is aninteger of 1-4, R³ is C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, C₁-C₄ alkoxy,phenyl optionally having substituent(s), or heteroaryl optionally havingsubstituent(s), a pharmaceutically acceptable salt thereof or a hydratethereof; and a pharmaceutically acceptable polymer. In one suchembodiment, the thienotriazolodiazepine compound is formulated as asolid dispersion comprising an amorphous thienotriazolodiazepinecompound.

In one embodiment, Formula (1A) is selected from the group consistingof: (i)(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamideor a dihydrate thereof, (ii) methyl(S)-{4-(3′-cyanobiphenyl-4-yl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]tri-azolo[4,3-a][1,4]diazepin-6-yl}acetate,(iii) methyl(S)-{2,3,9-trimethyl-4-(4-phenylaminophenyl)-6H-thieno[3,2-f][1,2,4]triaz-olo[4,3-a][1,4]diazepin-6-yl}acetate;and (iv) methyl(S)-{2,3,9-trimethyl-4-[4-(3-phenylpropionylamino)phenyl]-6H-thieno[3,2-f-][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate.In one such embodiment, Formula (1) is(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamide.

In some embodiments, the pharmaceutically acceptable polymer ishydroxypropylmethylcellulose acetate succinate. In some suchembodiments, the solid dispersion has a thienotriazolodiazepine compoundto hydroxypropylmethylcellulose acetate succinate (HPMCAS), weight ratioof 1:3 to 1:1. In some such embodiments, the solid dispersion exhibits asingle glass transition temperature (Tg) inflection point ranging fromabout 130° C. to about 140° C. In some such embodiments, a concentrationof the thienotriazolodiazepine compound after exposure to the relativehumidity of 75% at 40° C. for at least one month is at least 90% of theconcentration the amorphous thienotriazolodiazepine compound prior tosuch exposure.

In other embodiments, the pharmaceutically acceptable polymer is PVP. Insome such embodiments, the solid dispersion has athienotriazolodiazepine compound to PVP weight ratio of 1:3 to 1:1. Insome such embodiments, the solid dispersion exhibits a single glasstransition temperature (Tg) inflection point ranging from about 175° C.to about 185° C. In some such embodiments, a concentration of thethienotriazolodiazepine compound after exposure to the relative humidityof 75% at 40° C. for at least one month is at least 90% of theconcentration the amorphous thienotriazolodiazepine compound prior tosuch exposure.

In another embodiment, the solid dispersion is obtained by spray drying.

In another embodiment, the solid dispersion exhibits an X-ray powderdiffraction pattern substantially free of diffraction lines associatedwith crystalline thienotriazolodiazepine compound of Formula (1).

In yet another embodiment, the solid dispersion provides an area underthe curve (AUC) value that is at least 0.5 times that of a correspondingAUC value provided by a control composition administered intravenously,wherein the control composition comprises an equivalent quantity of acrystalline thienotriazolodiazepine compound of Formula (1).

In still yet another embodiment, the solid dispersion provides aconcentration, of the amorphous thienotriazolodiazepine compound, in anaqueous in vitro test medium at pH between 5.0 to 7.0, of at least5-fold greater than a concentration of a crystallinethienotriazolodiazepine compound of Formula (1) without polymer, in acontrol in vitro test medium at pH between 5.0 to 7.0 test medium.

In yet another embodiment, a concentration of the amorphousthienotriazolodiazepine compound, from the solid dispersion, in anaqueous in vitro test medium having a pH of 1.0 to 2.0, is at least 50%higher than a concentration of a crystalline thienotriazolodiazepinecompound of Formula (1) without polymer in an in vitro test mediumhaving a pH between 5.0 and 7.0.

In one embodiment, the concentration of the amorphousthienotriazolodiazepine compound, is at least 50% higher compared to aconcentration of thienotriazolodiazepine compound of Formula (1), from asolid dispersion of thienotriazolodiazepine compound of the Formula (1)and a pharmaceutically acceptable polymer selected from the groupconsisting of: hypromellose phthalate and ethyl acrylate-methylmethacrylate-trimethylammonioethyl methacrylate chloride copolymer,wherein each solid dispersion was placed in an aqueous in vitro testmedium having a pH of 1.0 to 2.0.

In one embodiment, the concentration of the amorphousthienotriazolodiazepine compound of Formula (1), is at least 50% highercompared to a concentration of thienotriazolodiazepine compound ofFormula (1), from a solid dispersion of thienotriazolodiazepine compoundof the Formula (1) and a pharmaceutically acceptable polymer selectedfrom the group consisting of: hypromellose phthalate and ethylacrylate-methyl methacrylate-trimethylammonioethyl methacrylate chloridecopolymer, wherein each solid dispersion was placed in an aqueous invitro test medium having a pH of 1.0 to 2.0.

The present disclosure further provides for a pharmaceutical formulationcomprising a spray dried solid dispersion, as described herein, and oneor more pharmaceutically acceptable excipients selected from the groupconsisting of: lactose monohydrate; microcrystalline cellulose;

croscarmellose sodium; colloidal silicon dioxide; magnesium stearate;and combinations thereof. In some embodiments, the pharmaceuticalformulation has a bulk density ranging from 0.55 g/cc to 0.60 g/cc. Insome embodiments, the pharmaceutical formation may be a pharmaceuticalcapsule. In some embodiments, the pharmaceutical formation may be apharmaceutical tablet.

The present disclosure further provides for a pharmaceutical formulationcomprising 10-15 wt. % of a spray dried solid dispersion, as describedherein, and hydroxypropylmethylcellulose acetate succinate (HPMCAS),wherein the thienotriazolodiazepine compound is amorphous in thedispersion and has a thienotriazolodiazepine compound tohydroxypropylmethylcellulose acetate succinate (HPMCAS), weight ratio of1:3 to 1:1; 45-50 wt. % of lactose monohydrate; 35-40 wt. % ofmicrocrystalline cellulose; 4-6 wt. % of croscarmellose sodium; 0.8-1.5wt. % of colloidal silicon dioxide; and 0.8-1.5 wt. % of magnesiumstearate.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofembodiments of the pharmaceutical compositions includingthienotriazolodiazepine formulations and methods of the presentinvention, will be better understood when read in conjunction with theappended drawings of exemplary embodiments. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown.

In the drawings:

The term OTX015 is synonymous with compound (1-1) throughout thedescription and drawings.

FIG. 1A illustrates dissolution profile of a comparator formulationcomprising a solid dispersion comprising 25% compound (1-1) and EudragitL100-55.

FIG. 1B illustrates dissolution profile of a comparator formulationcomprising a solid dispersion comprising 50% compound (1-1) and EudragitL100-55.

FIG. 1C illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 25% compound (1-1) andpolyvinylpyrrolidone (PVP).

FIG. 1D illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 50% compound (1-1) and PVP.

FIG. 1E illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 25% compound (1-1) andPVP-vinyl acetate (PVP-VA).

FIG. 1F illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 50% compound (1-1) and PVP-VA.

FIG. 1G illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 25% compound (1-1) andhypromellose acetate succinate (HPMCAS-M).

FIG. 1H illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 50% compound (1-1) andHPMCAS-M.

FIG. 1I illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 25% compound (1-1) andhypromellose phthalate (HPMCP-HP55).

FIG. 1J illustrates dissolution profile of an exemplary formulationcomprising a solid dispersion comprising 50% compound (1-1) andHMCP-HP55.

FIG. 2A illustrates results of in vivo screening of an exemplaryformulation comprising a solid dispersion of 25% compound (1-1) and PVP.

FIG. 2B illustrates results of an in vivo screening of an exemplaryformulation comprising a solid dispersion of 25% compound (1-1) andHPMCAS-M.

FIG. 2C illustrates results of an in vivo screening of an exemplaryformulation comprising a solid dispersion of 50% compound (1-1) andHPMCAS-M.

FIG. 3 illustrates powder X-ray diffraction profiles of soliddispersions of compound (1-1).

FIG. 4A illustrates modified differential scanning calorimetry trace fora solid dispersion of 25% compound (1-1) and PVP equilibrated underambient conditions.

FIG. 4B illustrates modified differential scanning calorimetry trace fora solid dispersion of 25% compound (1-1) and HPMCAS-M equilibrated underambient conditions.

FIG. 4C illustrates modified differential scanning calorimetry trace fora solid dispersion of 50% compound (1-1) and HPMCAS-M equilibrated underambient conditions.

FIG. 5 illustrates plot of glass transition temperature (Tg) versusrelative humidity (RH) for solid dispersions of 25% compound (1-1) andPVP or HMPCAS-M and 50% compound (1-1) and HPMCAS-MG.

FIG. 6 illustrates modified differential scanning calorimetry trace fora solid dispersion of 25% compound (1-1) and PVP equilibrated under 75%relative humidity.

FIGS. 7A and 7B illustrate plasma concentration versus time curves forCompound (1-1) after 1 mg/kg intravenous dosing (solid rectangles) and 3mg/kg oral dosing as 25% Compound (1-1):PVP (open circles), 25% Compound(1-1):HPMCAS-MG (open triangles), and 50% Compound (1-1):HPMCAS-MG (openinverted triangles). The inset depicts the same data plotted on asemilogarithmic scale.

FIGS. 8A and 8B illustrate plasma concentration versus time curves forCompound (1-1) after 3 mg/kg oral dosing as 25% Compound (1-1):PVP (opencircles), 25% Compound (1-1):HPMCAS-MG (open triangles), and 50%Compound (1-1):HPMCAS-MG (open inverted triangles). The inset depictsthe same data plotted on a semi-logarithmic scale.

FIG. 9 illustrates a powder X-ray diffraction profile of soliddispersions of compound (1-1) in HPMCAS-MG at time zero of a stabilitytest.

FIG. 10 illustrates a powder X-ray diffraction profile of soliddispersions of compound (1-1) in HPMCAS-MG after 1 month at 40° C. and75% relative humidity.

FIG. 11 illustrates a powder X-ray diffraction profile of soliddispersions of compound (1-1) in HPMCAS-MG after 2 months at 40° C. and75% relative humidity.

FIG. 12 illustrates a powder X-ray diffraction profile of soliddispersions of compound (1-1) in HPMCAS-MG after 3 month at 40° C. and75% relative humidity.

FIG. 13 illustrates the compound (1-1) antiproliferative effects after72 h and characterization of common mutations found in NSCLC cells.

FIG. 14 illustrates the reduction of the S phase cell fraction after 24h exposure to compound (1-1).

FIG. 15A illustrates BRDs, C-MYC, N-MYC, BCL2 and P21 mRNA levels inNSCLC cells classified in order of compound (1-1) sensitivity (GI₅₀values).

FIG. 15B illustrates basal protein expression of BRD2, BRD3, C-MYC,N-MYC, P21, BCL2 and cyclin D1 in NSCLC cells.

FIG. 16A illustrates C-MYC mRNA levels in HOP62, HOP92, H2228, H3122,and A549 cells after exposure to 500 nM of compound (1-1).

FIG. 16B illustrates N-MYC mRNA levels in HOP62, HOP92, H2228, H3122,and A549 cells after exposure to 500 nM of compound (1-1).

FIG. 16C illustrates regulation of C-MYC protein and N-MYC protein inHOP62, HOP92, H2228, H3122, and A549 cells after exposure to 500 nM ofcompound (1-1).

FIG. 17 illustrates the effects of compound (1-1) in concomitantcombination with everolimus or critzotinib in NSCLC cell lines.

FIG. 18A illustrates that compound (1-1) slowed tumor progression inH3122 tumor-bearing mice compared to vehicle control treated mice,crizotinib.

FIG. 18B illustrates that no weight loss was observed during the 3 weeksof compound (1-1) treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present subject matter will now be described more fully hereinafterwith reference to the accompanying Figures and Examples, in whichrepresentative embodiments are shown. The present subject matter can,however, be embodied in different forms and should not be construed aslimited to the embodiments set forth herein. Rather, these embodimentsare provided to describe and enable one of skill in the art. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the subject matter pertains. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entireties.

I. DEFINITIONS

The term “alkyl group” as used herein refers to a saturated straight orbranched hydrocarbon.

The term “substituted alkyl group” refers to an alkyl moiety having oneor more substituents replacing a hydrogen or one or more carbons of thehydrocarbon backbone.

The term “alkenyl group” whether used alone or as part of a substituentgroup, for example, “C1-4alkenyl(aryl),” refers to a partiallyunsaturated branched or straight chain monovalent hydrocarbon radicalhaving at least one carbon-carbon double bond, whereby the double bondis derived by the removal of one hydrogen atom from each of two adjacentcarbon atoms of a parent alkyl molecule and the radical is derived bythe removal of one hydrogen atom from a single carbon atom. Atoms may beoriented about the double bond in either the cis (Z) or trans (E)conformation. Typical alkenyl radicals include, but are not limited to,ethenyl, propenyl, allyl(2-propenyl), butenyl and the like. Examplesinclude C2-8alkenyl or C2-4alkenyl groups.

The term “C(j-k)” (where j and k are integers referring to a designatednumber of carbon atoms) refers to an alkyl, alkenyl, alkynyl, alkoxy orcycloalkyl radical or to the alkyl portion of a radical in which alkylappears as the prefix root containing from j to k carbon atomsinclusive. For example, C(1-4) denotes a radical containing 1, 2, 3 or 4carbon atoms.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The term “pharmaceutically acceptable salts” is art-recognized andrefers to the relatively non-toxic, inorganic and organic acid additionsalts, or inorganic or organic base addition salts of compounds,including, for example, those contained in compositions of the presentinvention.

The term “solid dispersion” as used herein refers to a group of solidproducts consisting of at least two different components, generally ahydrophilic carrier and a hydrophobic drug (active ingredient).

The term “chiral” is art-recognized and refers to molecules That havethe property of nonsuperimposability of the mirror image partner, whilethe term “achiral” refers to molecules which are superimposable on theirmirror image partner. A “prochiral molecule” is a molecule that has thepotential to be converted to a chiral molecule in a particular process.

The symbol “ ” is used to denote a bond that may be a single, a doubleor a triple bond.

The term “enantiomer” as it used herein, and structural formulasdepicting an enantiomer are meant to include the “pure” enantiomer freefrom its optical isomer as well as mixtures of the enantiomer and itsoptical isomer in which the enantiomer is present in an enantiomericexcess, e.g., at least 10%, 25%, 50%, 75%, 90%, 95%, 98%, or 99%enantiomeric excess.

The term “stereoisomers” when used herein consist of all geometricisomers, enantiomers or diastereomers. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Conformational isomers and rotamers of disclosed compounds are alsocontemplated.

The term “stereoselective synthesis” as it is used herein denotes achemical or enzymatic reaction in which a single reactant forms anunequal mixture of stereoisomers during the creation of a newstereocenter or during the transformation of a pre-existing one, and arewell known in the art. Stereoselective syntheses encompass both enantioselective and diastereoselective transformations. 15 For examples, seeCarreira, E. M. and Kvaerno, L., Classics in Stereoselective Synthesis,Wiley-VCH: Weinheim, 2009.

The term “spray drying” refers to processes which involve theatomization of the feed suspension or solution into small droplets andrapidly removing solvent from the mixture in a processor chamber wherethere is a strong driving force for the evaporation (e.g., hot dry gasor partial vacuum or combinations thereof).

The term “therapeutically effective amount” as used herein refers to anyamount of a thienotriazolodiazepine of the present invention or anyother pharmaceutically active agent which, as compared to acorresponding a patient who has not received such an amount of thethienotriazolodiazepine or the other pharmaceutically active agent,results in improved treatment, healing, prevention, or amelioration of adisease, disorder, or side effect, or a decrease in the rate ofadvancement of a disease or disorder.

The term “about” means+/−10%.

Throughout this application and in the claims that follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, should be understood to imply the inclusionof a stated integer step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

It has now been found that thienotriazolodiazepine compound of Formula(1), described herein below, can be formulated as a solid dispersionwith pharmaceutically acceptable polymers, to provide an oralformulation that provides high absorption of the pharmaceuticalingredient into the circulation from the gastrointestinal tract. In oneembodiment, the pharmaceutically acceptable polymer is hypromelloseacetate succinate (also called hydroxypropylmethylcellulose acetatesuccinate or HPMCAS). In one embodiment, the pharmaceutically acceptablepolymer is polyvinylpyrrolidone (PVP).

In some embodiments, the hydroxypropylmethyl cellulose acetatesuccinates (HPMCAS), may include M grade having 9% acetyl/11% succinoyl(e.g., HPMCAS having a mean particle size 10 of 5 μm (i.e., HPMCAS-MF,fine powder grade) or having a mean particle size of 1 mm (i.e.,HPMCAS-MG, granular grade)), H grade having 12% acetyl/6% succinoyl(e.g., HPMCAS having a mean particle size of 5 μm (i.e., HPMCAS-HF, finepowder grade) or having a mean particle size of 1 mm (i.e., HPMCAS-HG,granular grade)), and L grade having 8% acetyl/15% succinoyl (e.g.,HPMCAS having a mean particle size of 5 μm (i.e., HPMCAS-LF, fine powdergrade) or having a 15 mean particle size of 1 mm (i.e., HPMCAS-LG,granular grade).

In some embodiments, the polyvinyl pyrrolidones may have molecularweights of about 2,500 (Kollidon®12 PF, weight-average molecular weightbetween 2,000 to 3,000), about 9,000 (Kollidon® 17 PF, weight-averagemolecular weight between 7,000 to 11,000), about 25,000 (Kollidon® 25,weight-average molecular weight between 28,000 to 34,000), about 50,000(Kollidon® 30, weight-average molecular weight between 44,000 to54,000), and about 1,250,000 (Kollidon® 90 or Kollidon® 90F,weight-average molecular weight between 1,000,000 to 1,500,000).

II. METHODS OF TREATMENT

In one embodiment, the present disclosure provides for a method oftreating non-small cell 25 lung cancer in a mammal comprising:administering to a patient in need a pharmaceutically acceptable amountof a composition comprising a solid dispersion according to thecompositions described in sections III, IV, V, VI and VII. In oneembodiment, the non-small cell lung cancer is EML4-ALk positive. In onesuch embodiment, the non-small cell lung cancer exhibited downregulation of N-MYC mRNA levels after treatment. In another suchembodiment, the non-small cell lung cancer expressed BRD4/3/2, c-MYC,BCL-2, p21 and CyclinD1. In still another embodiment, the treatmentinduced a transient up-regulation of STAT3 with a subsequentdown-regulation after 24 hour and up to 72 hour exposure.

In some embodiments the method further comprises administering to thepatient an mTOR inhibitor. In one such embodiment the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus,ridaforolimus and everolimus. In one such embodiment the mTOR inhibitoris everolimus. The thienotriazolodiazepine compound and the mTORinhibitor can be administered simultaneously or sequentially. In someembodiments such combination of thienotrizolodiazepine compound and mTORinhibitor produces a synergistic effect.

In some embodiments the method further comprises administering to thepatient an ALK inhibitor. In one such embodiment the ALK inhibitor isselected from the group consisting of ceritinib and crizotinib. In onesuch embodiment the ALK inhibitor is crizotinib. Thethienotriazolodiazepine compound and the ALK inhibitor can beadministered simultaneously or sequentially. In some embodiments suchcombination of thienotrizolodiazepine compound and ALK inhibitorproduces a synergistic effect.

In still another embodiment, the non-small cell lung cancer is EML4-ALknegative. In one such embodiment, the non-small cell lung cancerexpressed BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1. In another suchembodiment, the treatment induced a transient up-regulation of STAT3with a subsequent down-regulation after 24 hour and up to 72 hourexposure.

In one embodiment, the non-small-cell lung cancer has a mutation in theKRAS gene. In one embodiment the non-small-cell lung cancer has amutation in the LKB1 gene. In some embodiments the non-small-cell lungcancer has a mutation in both the KRAS and LKB1 genes.

In one embodiment NMYC is downregulated. In one embodiment HEXIM isupregulated.

In some embodiments, methods use thienotriazolodiazepine compound of theFormula (1)

wherein

R¹ is alkyl having a carbon number of 1-4, R² is a hydrogen atom; ahalogen atom; or alkyl having a carbon number of 1-4 optionallysubstituted by a halogen atom or a hydroxyl group, R³ is a halogen atom;phenyl optionally substituted by a halogen atom, alkyl having a carbonnumber of 1-4, alkoxy having a carbon number of 1-4 or cyano;—NR⁵—(CH₂)_(m)—R⁶ wherein R⁵ is a hydrogen atom or alkyl having a carbonnumber of 1-4, m is an integer of 0-4, and R⁶ is phenyl or pyridyloptionally substituted by a halogen atom; or —NR⁷—CO—(CH₂)_(n)—R⁸wherein R⁷ is a hydrogen atom or alkyl having a carbon number of 1-4, nis an integer of 0-2, and R⁸ is phenyl or pyridyl optionally substitutedby a halogen atom, and R⁴ is —(CH₂)_(a)—CO—NH—R⁹ wherein a is an integerof 1-4, and R⁹ is alkyl having a carbon number of 1-4; hydroxyalkylhaving a carbon number of 1-4; alkoxy having a carbon number of 1-4; orphenyl or pyridyl optionally substituted by alkyl having a carbon numberof 1-4, alkoxy having a carbon number of 1-4, amino or a hydroxyl groupor —(CH₂)_(b)—COOR¹⁰ wherein b is an integer of 1-4, and R¹⁰ is alkylhaving a carbon number of 1-4, including any salts, isomers,enantiomers, racemates, hydrates, solvates, metabolites, and polymorphsthereof.

In some embodiments, Formula (1) is selected from Formula (1A):

wherein X is a halogen, R¹ is C₁-C₄ alkyl, R² is C₁-C₄ alkyl, a is aninteger of 1-4, R³ is C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, C₁-C₄ alkoxy,phenyl optionally having substituent(s), or heteroaryl optionally havingsubstituent(s), a pharmaceutically acceptable salt thereof or a hydratethereof; and a pharmaceutically acceptable polymer. In one suchembodiment, the thienotriazolodiazepine compound is formulated as asolid dispersion comprising an amorphous thienotriazolodiazepinecompound.

III. THIENOTRIAZOLODIAZEPINE COMPOUNDS

In one embodiment, the thienotriazolodiazepine compounds, used in theformulations of the present invention, are represented by Formula (1):

wherein R¹ is alkyl having a carbon number of 1-4, R² is a hydrogenatom; a halogen atom; or alkyl having a carbon number of 1-4 optionallysubstituted by a halogen atom or a hydroxyl group, R³ is a halogen atom;phenyl optionally substituted by a halogen atom, alkyl having a carbonnumber of 1-4, alkoxy having a carbon number of 1-4 or cyano;—NR⁵—(CH₂)_(m)—R⁶ wherein R⁵ is a hydrogen atom or alkyl having a carbonnumber of 1-4, m is an integer of 0-4, and R⁶ is phenyl or pyridyloptionally substituted by a halogen atom; or —NR⁷—CO—(CH₂)_(n)—R⁸wherein R⁷ is a hydrogen atom or alkyl having a carbon number of 1-4, nis an integer of 0-2, and R⁸ is phenyl or pyridyl optionally substitutedby a halogen atom, and R⁴ is —(CH₂)_(a)—CO—NH—R⁹ wherein a is an integerof 1-4, and R⁹ is alkyl having a carbon number of 1-4; hydroxyalkylhaving a carbon number of 1-4; alkoxy having a carbon number of 1-4; orphenyl or pyridyl optionally substituted by alkyl having a carbon numberof 1-4, alkoxy having a carbon number of 1-4, amino or a hydroxyl groupor —(CH₂)_(b)—COOR¹⁰ wherein b is an integer of 1-4, and R¹⁰ is alkylhaving a carbon number of 1-4, including any salts, isomers,enantiomers, racemates, hydrates, solvates, metabolites, and polymorphsthereof.

In one embodiment, a suitable alkyl group includes linear or branchedakyl radicals including from 1 carbon atom up to 4 carbon atoms. In oneembodiment, a suitable alkyl group includes linear or branched akylradicals including from 1 carbon atom up to 3 carbon atoms. In oneembodiment, a suitable alkyl group includes linear or branched akylradicals include from 1 carbon atom up to 2 carbon atoms. In oneembodiment, exemplary alkyl radicals include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl. In one embodiment, exemplary alkyl groups include, but arenot limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, and2-methyl-2-propyl.

In some embodiments, the present invention provides pharmaceuticallyacceptable salts, solvates, including hydrates, and isotopically-labeledforms of the thienotriazolodiazepine compounds described herein. In oneembodiment, pharmaceutically acceptable salts of thethienotriazolodiazepine compounds include acid addition salts formedwith inorganic acids. In one embodiment, pharmaceutically acceptableinorganic acid addition salts of the thienotriazolodiazepine includesalts of hydrochloric, hydrobromic, hydroiodic, phosphoric,metaphosphoric, nitric and sulfuric acids. In one embodiment,pharmaceutically acceptable salts of the thienotriazolodiazepinecompounds include acid addition salts formed with organic acids. In oneembodiment, pharmaceutically acceptable organic acid addition salts ofthe thienotriazolodiazepine include salts of tartaric, acetic,trifluoroacetic, citric, malic, lactic, fumaric, benzoic, formic,propionic, glycolic, gluconic, maleic, succinic, camphorsulfuric,isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic,furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,pantothenic, stearic, sulfinilic, alginic, galacturonic andarylsulfonic, for example benzenesulfonic and 4-methyl benzenesulfonicacids.

Representative thienotriazolodiazepine compounds of Formula (1) include,but are not limited to, the thienotriazolodiazepine compounds (1-1) to(1-18), which are listed in the following Table A.

Compound (1-1), of Table A, will be referred to herein as OTX-015,OTX015 or Y803.

TABLE A Exemplary compounds of the invention:

(1-1)

(1-2)

(1-3)

(1-4)

(1-5)

(1-6)

(1-7)

(1-8)

(1-9)

(1-10)

(1-11)

(1-12)

(1-13)

(1-14)

(1-15)

(1-16)

(1-17)

(1-18)

In some embodiments, thienotriazolodiazepine compounds of Formula (1)include (i)(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamideor a dihydrate thereof, (ii) methyl(S)-{4-(3′-cyanobiphenyl-4-yl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]tri-azolo[4,3-a][1,4]diazepin-6-yl}acetate,(iii) methyl(S)-{2,3,9-trimethyl-4-(4-phenylaminophenyl)-6H-thieno[3,2-f][1,2,4]triaz-olo[4,3-a][1,4]diazepin-6-yl}acetate;and (iv) methyl(S)-{2,3,9-trimethyl-4-[4-(3-phenylpropionylamino)phenyl]-6H-thieno[3,2-f-][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate.

In some embodiments, thienotriazolodiazepine compounds of Formula (1)include(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate.

In some embodiments, thienotriazolodiazepine compounds of Formula (1)include(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamide.

IV. FORMULATIONS

The compound of Formula (1) presents highly specific difficulties inrelation to administration generally and the preparation of galeniccompositions in particular, including the particular problems of drugbioavailability and variability in inter- and intra-patient doseresponse, necessitating development of a non-conventional dosage formwith respect to the practically water-insoluble properties of thecompound.

Previously, it had been found that the compound of Formula (1) could beformulated as a solid dispersion with the carrier ethyl acrylate-methylmethacrylate-trimethylammonioethyl methacrylate chloride copolymer(Eudragit RS, manufactured by Rohm) to provide an oral formulation thatpreferentially released the pharmaceutical ingredient in the lowerintestine for treatment of inflammatory bowel diseases such asulcerative colitis and Crohn's disease (US Patent Application20090012064 A1, published Jan. 8, 2009). It was found, through variousexperiments, including animal tests, that in inflammatory bowel diseasesdrug release in a lesion and a direct action thereof on the inflammatorylesion were more important than the absorption of the drug intocirculation from the gastrointestinal tract.

It has now been unexpectedly found that thienotriazolodiazepinecompounds, according to Formula (1), pharmaceutically acceptable salts,solvates, including hydrates, racemates, enantiomers isomers, andisotopically-labeled forms thereof, can be formulated as a soliddispersion with pharmaceutically acceptable polymers to provide an oralformulation that provides high absorption of the pharmaceuticalingredient into the circulation from the gastrointestinal tract fortreatment of diseases other than inflammatory bowel diseases. Studies inboth dogs and humans have confirmed high oral bioavailability of thesesolid dispersions compared with the Eudragit solid dispersionformulation previously developed for the treatment of inflammatory boweldisease.

Solid dispersions are a strategy to improve the oral bioavailability ofpoorly water soluble drugs.

The term “solid dispersion” as used herein refers to a group of solidproducts including at least two different components, generally ahydrophilic carrier and a hydrophobic drug, the thienotriazolodiazepinecompounds, according to Formula (1). Based on the drug's moleculararrangement within the dispersion, six different types of soliddispersions can be distinguished. Commonly, solid dispersions areclassified as simple eutectic mixtures, solid solutions, glass solutionand suspension, and amorphous precipitations in a crystalline carrier.Moreover, certain combinations can be encountered, for example, in thesame sample some molecules may be present in clusters while some aremolecularly dispersed.

In one embodiment, the thienotriazolodiazepine compounds, according toFormula (1) can be dispersed molecularly, in amorphous particles(clusters). In another embodiment, the thienotriazolodiazepinecompounds, according to Formula (1) can be dispersed as crystallineparticles. In one embodiment, the carrier can be crystalline. In anotherembodiment, the carrier can be amorphous.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a solid dispersion of a thienotriazolodiazepinecompound, in accordance with Formula (1), or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof; and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is hypromellose acetate succinate(also called hydroxypropylmethylcellulose acetate succinate or HPMCAS).In one embodiment, the dispersion has a thienotriazolodiazepine compoundto hydroxypropylmethylcellulose acetate succinate (HPMCAS) weight ratioof 1:3 to 1:1. In one embodiment, at least some portion of thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In another embodiment, the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Insome embodiments, the solid dispersion exhibits a single inflection forthe glass transition temperature (Tg). In some embodiments, the singleTg occurs between 130° C. to 140° C. In other such embodiments, thesingle Tg occurs at about 135° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1). In some embodiments, the hydroxypropylmethyl celluloseacetate succinates (HPMCAS), may include M grade having 9% acetyl/11%succinoyl (e.g., HPMCAS having a mean particle size of 5 μm (i.e.,HPMCAS-MF, fine powder grade) or having a mean particle size of 1 mm(i.e., HPMCAS-MG, granular grade)), H grade having 12% acetyl/6%succinoyl (e.g., HPMCAS having a mean particle size of 5 μm (i.e.,HPMCAS-HF, fine powder grade) or having a mean particle size of 1 mm(i.e., HPMCAS-HG, granular grade)), and L grade having 8% acetyl/15%succinoyl (e.g., HPMCAS having a mean particle size of 5 μm (i.e.,HPMCAS-LF, fine powder grade) or having a mean particle size of 1 mm(i.e., HPMCAS-LG, granular grade).

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a solid dispersion of a thienotriazolodiazepinecompound of Formula (1) or a pharmaceutically acceptable salt, asolvate, including a hydrate, a racemate, an enantiomer, an isomer, oran isotopically-labeled form thereof in a pharmaceutically acceptablepolymer. In one embodiment, the pharmaceutically acceptable polymer ispolyvinylpyrrolidone (also called povidone or PVP). In one embodiment,the dispersion has a thienotriazolodiazepine compound to PVP weightratio of 1:3 to 1:1. In one embodiment, at least some portion of thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In another embodiment, the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Insome embodiments, the solid dispersion exhibits a single inflection forthe glass transition temperature (Tg). In some embodiments, the singleTg occurs between 175° C. to about 185° C. In other such embodiments,the single Tg occurs at about 179° C. In some such embodiments, thesolid dispersion was exposed to a relative humidity of 75% at 40° C. forat least one month. In some embodiments, the solid dispersion exhibitsan X-ray powder diffraction pattern substantially free of diffractionlines associated with crystalline thienotriazolodiazepine compound ofFormula (1). For the purpose of this application “substantially free”shall mean the absence of a diffraction line, above the amorphous halo,at about 21° 2-theta associated with crystalline thienotriazolodiazepinecompound of Formula (1). In some embodiments, the polyvinyl pyrrolidonesmay have molecular weights of about 2,500 (Kollidon®12 PF,weight-average molecular weight between 2,000 to 3,000), about 9,000(Kollidon® 17 PF, weight-average molecular weight between 7,000 to11,000), about 25,000 (Kollidon® 25, weight-average molecular weightbetween 28,000 to 34,000), about 50,000 (Kollidon® 30, weight-averagemolecular weight between 44,000 to 54,000), and about 1,250,000(Kollidon® 90 or Kollidon® 90F, weight-average molecular weight between1,000,000 to 1,500,000).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of an amorphous form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is hypromellose acetate succinate.In one embodiment, the weight ratio of thienotriazolodiazepine compoundof Formula (1) to hypromellose acetate succinate ranges from 1:3 to 1:1.In one embodiment, at least some portion of the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inanother embodiment, the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 130° C. to 140° C. In other such embodiments, the singleTg occurs at about 135° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of an amorphous form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is polyvinylpyrrolidone. In oneembodiment, the weight ratio of thienotriazolodiazepine compound ofFormula (1) to polyvinylpyrrolidone ranges from 1:3 to 1:1. In oneembodiment, at least some portion of the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inanother embodiment, the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 175° C. to about 185° C. In other such embodiments, thesingle Tg occurs at about 179° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of a crystalline form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is hypromellose acetate succinate.In one embodiment, the weight ratio of thienotriazolodiazepine compoundof Formula (1) to hypromellose acetate succinate ranges from 1:3 to 1:1.

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of a crystalline form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is polyvinylpyrrolidone. In oneembodiment, the weight ratio of thienotriazolodiazepine compound ofFormula (1) to polyvinylpyrrolidone ranges from 1:3 to 1:1.

In some embodiments, a pharmaceutical composition comprising a soliddispersion is prepared by spray drying.

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of a thienotriazolodiazepinecompound of Formula (1) or a pharmaceutically acceptable salt, asolvate, including a hydrate, a racemate, an enantiomer, an isomer, oran isotopically-labeled form thereof and a pharmaceutically acceptablepolymer. In one embodiment, the pharmaceutically acceptable polymer ishypromellose acetate succinate. In one embodiment, the weight ratio ofcompound (1) to hypromellose acetate succinate ranges from 1:3 to 1:1.In one embodiment, at least some portion of the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inanother embodiment, the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 130° C. to 140° C. In other such embodiments, the singleTg occurs at about 135° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of a thienotriazolodiazepinecompound of Formula (1) or a pharmaceutically acceptable salt, asolvate, including a hydrate, a racemate, an enantiomer, an isomer, oran isotopically-labeled form thereof and a pharmaceutically acceptablepolymer. In one embodiment, the pharmaceutically acceptable polymer ispolyvinylpyrrolidone. In one embodiment, the weight ratio of compound(1) to polyvinylpyrrolidone ranges from 1:3 to 1:1. In one embodiment,at least some portion of the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In anotherembodiment, the thienotriazolodiazepine compound is homogeneouslydispersed throughout the solid dispersion. In some embodiments, thesolid dispersion exhibits a single inflection for the glass transitiontemperature (Tg). In some embodiments, the single Tg occurs between 175°C. to 185° C. In other such embodiments, the single Tg occurs at about179° C. In some such embodiments, the solid dispersion was exposed to arelative humidity of 75% at 40° C. for at least one month. In someembodiments, the solid dispersion exhibits an X-ray powder diffractionpattern substantially free of diffraction lines associated withcrystalline thienotriazolodiazepine compound of Formula (1). For thepurpose of this application “substantially free” shall mean the absenceof a diffraction line, above the amorphous halo, at about 21° 2-thetaassociated with crystalline thienotriazolodiazepine compound of Formula(1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of an amorphous form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is hypromellose acetate succinate.In one embodiment, the weight ratio of thienotriazolodiazepine compoundof Formula (1) to hypromellose acetate succinate ranges from 1:3 to 1:1.In one embodiment, at least some portion of the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inanother embodiment, the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 130° C. to 140° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In other such embodiments, the single Tg occurs atabout 135° C. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of an amorphous form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is polyvinylpyrrolidone. In oneembodiment, the weight ratio of thienotriazolodiazepine compound ofFormula (1) to polyvinylpyrrolidone ranges from 1:3 to 1:1. In oneembodiment, at least some portion of the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inanother embodiment, the thienotriazolodiazepine compound ishomogeneously dispersed throughout the solid dispersion. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 175° C. to 185° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In other such embodiments, the single Tg occurs atabout 179° C. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound of Formula(1). For the purpose of this application “substantially free” shall meanthe absence of a diffraction line, above the amorphous halo, at about21° 2-theta associated with crystalline thienotriazolodiazepine compoundof Formula (1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of a crystalline form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is hypromellose acetate succinate.In one embodiment, the weight ratio of thienotriazolodiazepine compoundof Formula (1) to hypromellose acetate succinate ranges from 1:3 to 1:1.

In one embodiment, a pharmaceutical composition of the present inventioncomprises a spray dried solid dispersion of a crystalline form of athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof and apharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is polyvinylpyrrolidone. In oneembodiment, the weight ratio of thienotriazolodiazepine compound ofFormula (1) to polyvinylpyrrolidone ranges from 1:3 to 1:1.

In one preferred embodiment, the present invention provides apharmaceutical composition comprising a solid dispersion of2-[(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thienol[3,2-f]-[1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate, compound (1-1):

or a pharmaceutically acceptable salt, a solvate, including a hydrate, aracemate, an enantiomer, an isomer, or an isotopically-labeled form anda pharmaceutically acceptable polymer. In one embodiment, thepharmaceutically acceptable polymer is HPMCAS. In one embodiment, thedispersion has compound (1-1) and HPMCAS in a weight ratio of 1:3 to1:1. In one embodiment, at least some portion of thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In another embodiment, the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inone embodiment, the solid dispersion is spray dried. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 130° C. to 140° C. In other such embodiments, the singleTg occurs at about 135° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound (1-1). Forthe purpose of this application “substantially free” shall mean theabsence of a diffraction line, above the amorphous halo, at about 21°2-theta associated with crystalline thienotriazolodiazepine compound(1-1).

In another embodiment, the pharmaceutical composition comprises a soliddispersion compound (1-1) or a pharmaceutically acceptable salt, asolvate, including a hydrate, a racemate, an enantiomer, an isomer, oran isotopically-labeled form; and a pharmaceutically acceptable polymer.In one embodiment, the pharmaceutically acceptable polymer is PVP. Inone embodiment, the dispersion has compound (1-1) and PVP in a weightratio 1:3 to 1:1. In one embodiment, at least some portion of thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In another embodiment, the thienotriazolodiazepinecompound is homogeneously dispersed throughout the solid dispersion. Inone embodiment, the solid dispersion is spray dried. In someembodiments, the solid dispersion exhibits a single inflection for theglass transition temperature (Tg). In some embodiments, the single Tgoccurs between 175° C. to 185° C. In other such embodiments, the singleTg occurs at about 179° C. In some such embodiments, the soliddispersion was exposed to a relative humidity of 75% at 40° C. for atleast one month. In some embodiments, the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline thienotriazolodiazepine compound (1-1). Forthe purpose of this application “substantially free” shall mean theabsence of a diffraction line, above the amorphous halo, at about 21°2-theta associated with crystalline thienotriazolodiazepine compound(1-1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of an amorphous form of athienotriazolodiazepine compound (1-1) or a pharmaceutically acceptablesalt, a solvate, including a hydrate, a racemate, an enantiomer, anisomer, or an isotopically-labeled form thereof; and a pharmaceuticallyacceptable polymer. In one embodiment, the pharmaceutically acceptablepolymer is HPMCAS. In one embodiment, the dispersion has compound (1-1)and HPMCAS in a weight ratio of 1:3 to 1:1. In one embodiment, at leastsome portion of the thienotriazolodiazepine compound is homogeneouslydispersed throughout the solid dispersion. In another embodiment, thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In one embodiment, the solid dispersion is spraydried. In some embodiments, the solid dispersion exhibits a singleinflection for the glass transition temperature (Tg). In someembodiments, the single Tg occurs between 130° C. to 140° C. In othersuch embodiments, the single Tg occurs at about 135° C. In some suchembodiments, the solid dispersion was exposed to a relative humidity of75% at 40° C. for at least one month. In some embodiments, the soliddispersion exhibits an X-ray powder diffraction pattern substantiallyfree of diffraction lines associated with crystallinethienotriazolodiazepine compound (1-1). For the purpose of thisapplication “substantially free” shall mean the absence of a diffractionline, above the amorphous halo, at about 21° 2-theta associated withcrystalline thienotriazolodiazepine compound (1-1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of an amorphous form of athienotriazolodiazepine compound (1-1) or a pharmaceutically acceptablesalt, a solvate, including a hydrate, a racemate, an enantiomer, anisomer, or an isotopically-labeled form thereof; and a pharmaceuticallyacceptable polymer. In one embodiment, the pharmaceutically acceptablepolymer is PVP. In one embodiment, the dispersion has compound (1-1) andPVP in a weight ratio 1:3 to 1:1. In one embodiment, at least someportion of the thienotriazolodiazepine compound is homogeneouslydispersed throughout the solid dispersion. In another embodiment, thethienotriazolodiazepine compound is homogeneously dispersed throughoutthe solid dispersion. In one embodiment, the solid dispersion is spraydried. In some embodiments, the solid dispersion exhibits a singleinflection for the glass transition temperature (Tg). In someembodiments, the single Tg occurs between 175° C. to 185° C. In othersuch embodiments, the single Tg occurs at about 189° C. In some suchembodiments, the solid dispersion was exposed to a relative humidity of75% at 40° C. for at least one month. In some embodiments, the soliddispersion exhibits an X-ray powder diffraction pattern substantiallyfree of diffraction lines associated with crystallinethienotriazolodiazepine compound (1-1). For the purpose of thisapplication “substantially free” shall mean the absence of a diffractionline, above the amorphous halo, at about 21° 2-theta associated withcrystalline thienotriazolodiazepine compound (1-1).

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of a crystalline form of athienotriazolodiazepine compound (1-1) or a pharmaceutically acceptablesalt, a solvate, including a hydrate, a racemate, an enantiomer, anisomer, or an isotopically-labeled form thereof; and a pharmaceuticallyacceptable polymer. In one embodiment, the pharmaceutically acceptablepolymer is HPMCAS. In one embodiment, the dispersion has compound (1-1)and HPMCAS in a weight ratio of 1:3 to 1:1. In one embodiment, the soliddispersion is spray dried.

In one embodiment, a pharmaceutical composition of the present inventioncomprises a solid dispersion of a crystalline form of athienotriazolodiazepine compound (1-1) or a pharmaceutically acceptablesalt, a solvate, including a hydrate, a racemate, an enantiomer, anisomer, or an isotopically-labeled form thereof; and a pharmaceuticallyacceptable polymer. In one embodiment, the pharmaceutically acceptablepolymer is PVP. In one embodiment, the dispersion has compound (1-1) andPVP in a weight ratio 1:3 to 1:1. In one embodiment, the soliddispersion is spray dried.

The solid dispersions of the invention, described herein, exhibitespecially advantageous properties when administered orally. Examples ofadvantageous properties of the solid dispersions include, but are notlimited to, consistent and high level of bioavailability whenadministered in standard bioavailability trials in animals or humans.The solid dispersions of the invention can include a solid dispersioncomprising thienotriazolodiazepine compound of Formula (1) and a polymerand additives. In some embodiments, the solid dispersions can achieveabsorption of the thienotriazolodiazepine compound of Formula (1) intothe bloodstream that cannot be obtained by merely admixing thethienotriazolodiazepine compound of Formula (1) with additives since thethienotriazolodiazepine compound of Formula (1) drug has negligiblesolubility in water and most aqueous media. The bioavailability, ofthienotriazolodiazepine compound of Formula (1) or ofthienotriazolodiazepine compound (1-1) may be measured using a varietyof in vitro and/or in vivo studies. The in vivo studies may beperformed, for example, using rats, dogs or humans.

The bioavailability may be measured by the area under the curve (AUC)value obtained by plotting a serum or plasma concentration, of thethienotriazolodiazepine compound of Formula (1) orthienotriazolodiazepine compound (1-1), along the ordinate (Y-axis)against time along the abscissa (X-axis). The AUC value of thethienotriazolodiazepine compound of Formula (1) orthienotriazolodiazepine compound (1-1) from the solid dispersion, isthen compared to the AUC value of an equivalent concentration ofcrystalline thienotriazolodiazepine compound of Formula (1) orcrystalline thienotriazolodiazepine compound (1-1) without polymer. Insome embodiments, the solid dispersion provides an area under the curve(AUC) value, when administered orally to a dog, that is selected from:at least 0.4 times, 0.5 times, 0.6 time, 0.8 time, 1.0 times, acorresponding AUC value provided by a control composition administeredintravenously to a dog, wherein the control composition comprises anequivalent quantity of a crystalline thienotriazolodiazepine compound ofFormula I.

The bioavailability may be measured by in vitro tests simulating the pHvalues of a gastric environment and an intestine environment. Themeasurements may be made by suspending a solid dispersion of thethienotriazolodiazepine compound of Formula (1) orthienotriazolodiazepine compound (1-1), in an aqueous in vitro testmedium having a pH between 1.0 to 2.0, and the pH is then adjusted to apH between 5.0 and 7.0, in a control in vitro test medium. Theconcentration of the amorphous thienotriazolodiazepine compound ofFormula (1) or amorphous thienotriazolodiazepine compound (1-1) may bemeasured at any time during the first two hours following the pHadjustment. In some embodiments, the solid dispersion provides aconcentration, of the amorphous thienotriazolodiazepine compound ofFormula (1) or amorphous thienotriazolodiazepine compound (1-1), in anaqueous in vitro test medium at pH between 5.0 to 7.0 that is selectedfrom: at least 5-fold greater, at least 6 fold greater, at least 7 foldgreater, at least 8 fold greater, at least 9 fold greater or at least 10fold greater, compared to a concentration of a crystallinethienotriazolodiazepine compound of Formula (1) or crystallinethienotriazolodiazepine compound (1-1), without polymer.

In other embodiments, the concentration of the amorphousthienotriazolodiazepine compound of Formula (1) or amorphousthienotriazolodiazepine compound (1-1), from the solid dispersion placedin an aqueous in vitro test medium having a pH of 1.0 to 2.0, is: atleast 40%, at least 50% higher, at least 60%, at least 70%; at least80%, than a concentration of a crystalline thienotriazolodiazepinecompound of Formula (1) without polymer. In some such embodiments, thepolymer of the solid dispersion is HPMCAS. In some such embodiments, thepolymer of the solid dispersion is PVP.

In other embodiments, a concentration of the amorphousthienotriazolodiazepine compound of Formula (1) or amorphousthienotriazolodiazepine compound (1-1), from the solid dispersion, is:at least 40%, at least 50% higher, at least 60%, at least 70%; at least80%, compared to a concentration of thienotriazolodiazepine compound ofFormula (1), from a solid dispersion of thienotriazolodiazepine compoundof the Formula (1) and a pharmaceutically acceptable polymer selectedfrom the group consisting of: hypromellose phthalate and ethylacrylate-methyl methacrylate-trimethylammonioethyl methacrylate chloridecopolymer, wherein each solid dispersion was placed in an aqueous invitro test medium having a pH of 1.0 to 2.0. In some such embodiments,the polymer of the solid dispersion is HPMCAS. In some such embodiments,the polymer of the solid dispersion is PVP.

In some embodiments, the solid dispersions, described herein, exhibitstability against recrystallization of the thienotriazolodiazepinecompound of the Formula (1) or the thienotriazolodiazepine compound(1-1) when exposed to humidity and temperature over time. In oneembodiment, the concentration of the amorphous thienotriazolodiazepinecompound of the Formula (1) or the thienotriazolodiazepine compound(1-1) which remains amorphous is selected from: at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% and at least 99%.

V. DOSAGE FORMS

Suitable dosage forms that can be used with the solid dispersions of thepresent invention include, but are not limited to, capsules, tablets,mini-tablets, beads, beadlets, pellets, granules, granulates, andpowder. Suitable dosage forms may be coated, for example using anenteric coating. Suitable coatings may comprise but are not limited tocellulose acetate phthalate, hydroxypropylmethylcellulose (HPMC),hydroxypropylmethylcellulose phthalate, a polymethylacrylic acidcopolymer, or hydroxylpropylmethylcellulose acetate succinate (HPMCAS).In some embodiments, certain combinations can be encountered, forexample, in the same sample some molecules of thethienotriazolodiazepine of the present invention may be present inclusters while some are molecularly dispersed with a carrier.

In some embodiments, the solid dispersions of the invention may beformulated as tablets, caplets, or capsules. In one some embodiments,the solid dispersions of the invention may be formulated as mini-tabletsor pour-into-mouth granules, or oral powders for constitution. In someembodiments, the solid dispersions of the invention are dispersed in asuitable diluent in combination with other excipients (e.g.,re-crystallization/precipitation inhibiting polymers, taste-maskingcomponents, etc) to give a ready-to-use suspension formulation. In someembodiments, the solid dispersions of the invention may be formulatedfor pediatric treatment.

In one embodiment, the pharmaceutical composition of the presentinvention is formulated for oral administration. In one embodiment, thepharmaceutical composition comprises a solid dispersion, according tothe various embodiments described herein, comprising athienotriazolodiazepine compound of Formula (1) or a pharmaceuticallyacceptable salt, a solvate, including a hydrate, a racemate, anenantiomer, an isomer, or an isotopically-labeled form thereof; and apolymer carrier. In one embodiment, the pharmaceutical compositionfurther includes one or more additives such as disintegrants,lubricants, glidants, binders, and fillers.

Examples of suitable pharmaceutically acceptable lubricants andpharmaceutically acceptable glidants for use with the pharmaceuticalcomposition include, but are not limited to, colloidal silica, magnesiumtrisilicate, starches, talc, tribasic calcium phosphate, magnesiumstearate, aluminum stearate, calcium stearate, magnesium carbonate,magnesium oxide, polyethylene glycol, powdered cellulose, glycerylbehenate, stearic acid, hydrogenated castor oil, glyceryl monostearate,and sodium stearyl fumarate.

Examples of suitable pharmaceutically acceptable binders for use withthe pharmaceutical composition include, but are not limited to starches;celluloses and derivatives thereof, e.g., microcrystalline cellulose(e.g., AVICEL PH from FMC), hydroxypropyl cellulose, hydroxyethylcellulose, and hydroxylpropylmethylcellulose (HPMC, e.g., METHOCEL fromDow Chemical); sucrose, dextrose, corn syrup; polysaccharides; andgelatin.

Examples of suitable pharmaceutically acceptable fillers andpharmaceutically acceptable diluents for use with the pharmaceuticalcomposition include, but are not limited to, confectioner's sugar,compressible sugar, dextrates, dextrin, dextrose, lactose, mannitol,microcrystalline cellulose (MCC), powdered cellulose, sorbitol, sucrose,and talc.

In some embodiments, excipients may serve more than one function in thepharmaceutical composition. For example, fillers or binders may also bedisintegrants, glidants, anti-adherents, lubricants, sweeteners and thelike.

In some embodiments, the pharmaceutical compositions of the presentinvention may further include additives or ingredients, such asantioxidants (e.g., ascorbyl palmitate, butylated hydroxylanisole (BHA),butylated hydroxytoluene (BHT), α-tocopherols, propyl gallate, andfumaric acid), antimicrobial agents, enzyme inhibitors, stabilizers(e.g., malonic acid), and/or preserving agents.

Generally, the pharmaceutical compositions of the present invention maybe formulated into any suitable solid dosage form. In some embodiments,the solid dispersions of the invention are compounded in unit dosageform, e.g., as a capsule, or tablet, or a multi-particulate system suchas granules or granulates or a powder, for administration.

In one embodiment, a pharmaceutical compositions includes a soliddispersion of a thienotriazolodiazepine compound of Formula (1),according to the various embodiments of solid dispersions describedherein, and hydroxypropylmethylcellulose acetate succinate (HPMCAS),wherein the thienotriazolodiazepine compound is amorphous in the soliddispersion and has a thienotriazolodiazepine compound tohydroxypropylmethylcellulose acetate succinate (HPMCAS), weight ratio of1:3 to 1:1; 45-50 wt. % of lactose monohydrate; 35-40 wt. % ofmicrocrystalline cellulose; 4-6 wt. % of croscarmellose sodium; 0.8-1.5wt. % of colloidal silicon dioxide; and 0.8-1.5 wt. % of magnesiumstearate.

VI. DOSAGE

In one embodiment, the present invention provides a pharmaceuticalcomposition that maybe formulated into any suitable solid dosage form.In one embodiment, a pharmaceutical composition in accordance with thepresent invention comprises one or more of the various embodiments ofthe thienotriazolodiazepine of Formula (1) as described herein in adosage amount ranging from about 10 mg to about 100 mg. In oneembodiment, the pharmaceutical composition of the present inventionincludes one or more of the various embodiments of thethienotriazolodiazepine of Formula (1) as described herein in a dosageamount selected from the group consisting of from about 10 mg to about100 mg, about 10 mg to about 90 mg, about 10 mg to about 80 mg, about 10mg to about 70 mg, about 10 mg to about 60 mg, about 10 mg to about 50mg, about 10 mg to about 40 mg, about 10 mg to about 30 mg, and about 10mg to about 20 mg. In one embodiment, the pharmaceutical composition ofthe present invention includes one or more of the various embodiments ofthe thienotriazolodiazepine of Formula (1) as described herein in adosage amount selected from the group consisting of about 10 mg, about50 mg, about 75 mg, about 100 mg.

In some embodiments, the methods of the present invention includesadministering to a subject in need thereof one or more of the variousembodiments of the thienotriazolodiazepine of Formula (I) as describedherein in a dosage amount selected from the group consisting of about 1mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg,about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg,about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg,about 130 mg, about 140 mg, and about 150 mg, and in a dosage formselected from the group consisting of once weekly, once daily everysixth day, once daily every fifth day, once daily every fourth day, oncedaily every third day, once daily every other day, once daily, twicedaily, three times daily, four times daily, and five times daily. Inanother embodiment, any of the foregoing dosage amounts or dosage formsis decreased periodically or increased periodically.

In some embodiments, the methods of the present invention includesadministering to a subject in need thereof a thienotriazolodiazepineselected from the group consisting of compounds (1-1), (1-2), (1-3),(1-4), (1-5), (1-6), (1-7), (1-8), (1-9), (1-10), (1-11), (1- 12),(1-13), (1-14), (1-15), (1-16), (1-17), and (1-18), in a dosage amountselected from the group consisting of about 1 mg, about 2 mg, about 2.5mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg,about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg,about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg,and about 150 mg, and in a dosage form selected from the groupconsisting of once weekly, once daily every sixth day, once daily everyfifth day, once daily every fourth day, once daily every third day, oncedaily every other day, once daily, twice daily, three times daily, fourtimes daily, and five times daily. In another embodiment, any of theforegoing dosage amounts or dosage forms is decreased periodically orincreased periodically.

Such unit dosage forms are suitable for administration 1 to 5 timesdaily depending on the particular purpose of therapy, the phase oftherapy, and the like. In one embodiment, the dosage form may beadministered to a subject in need thereof at least once daily for atleast two successive days. In one embodiment, the dosage form may beadministered to a subject in need thereof at least once daily onalternative days. In one embodiment, the dosage form may be administeredto a subject in need thereof at least weekly and divided into equaland/or unequal doses. In one embodiment, the dosage form may beadministered to a subject in need thereof weekly, given either on threealternate days and/or 6 times per week. In one embodiment, the dosageform may be administered to a subject in need thereof in divided doseson alternate days, every third day, every fourth day, every fifth day,every sixth day and/or weekly. In one embodiment, the dosage form may beadministered to a subject in need thereof two or more equally orunequally divided doses per month.

The dosage form used, e.g., in a capsule, tablet, mini-tablet, beads,beadlets, pellets, granules, granulates, or powder may be coated, forexample using an enteric coating. Suitable coatings may comprise but arenot limited to cellulose acetate phthalate, hydroxypropylmethylcellulose(HPMC), hydroxypropylmethylcellulose phthalate, a polymethylacrylic acidcopolymer, or hydroxylpropylmethylcellulose acetate succinate (HPMCAS).

VII. PROCESS

The thienotriazolodiazepine compounds disclosed herein can exist as freebase or as acid addition salt can be obtained according to theprocedures described in US Patent Application Publication No.2010/0286127, incorporated by reference in its entirety herein, or inthe present application. Individual enantiomers and diastereomers of thethienotriazolodiazepine compounds of the present invention can beprepared synthetically from commercially available starting materialsthat contain asymmetric or stereogenic centers, or by preparation ofracemic mixtures followed by resolution methods well known to those ofordinary skill in the art.

In some embodiments, a one or more of the various embodiments for theformulation of the thienotriazolodiazepine, according to Formula (1), isprepared by a solvent evaporation method. In one embodiment, the solventevaporation method comprises solubilization of a thienotriazolodiazepinecompound, according to Formula (1), carrier in a volatile solvent thatis subsequently evaporated. In one embodiment, the volatile solvent mayone or more excipients. In one embodiment, the one or more excipientsinclude, but are not limited to anti-sticking agents, inert fillers,surfactants wetting agents, pH modifiers and additives. In oneembodiment, the excipients may dissolved or in suspended or swollenstate in the volatile solvent.

In one embodiment, preparation of solid dispersions in accordance withthe present invention includes drying one or more excipients suspendedin a volatile solvent. In one embodiment, the drying includes vacuumdrying, slow evaporation of the volatile solvent at low temperature, useof a rotary evaporator, spray-drying, spray granulation, freeze-drying,or use of supercritical fluids.

In one embodiment, spray drying preparation of a formulation for thethienotriazolodiazepine composition, according to Formula (1), is usedwhich involves atomization of a suspension or a solution of thecomposition into small droplets, followed by rapid removal solvent fromthe formulation. In one embodiment, preparation of a formulation inaccordance with the present invention involves spray granulation inwhich a solution or a suspension of the composition in a solvent issprayed onto a suitable chemically and/or physically inert filler, suchas lactose or mannitol. In one embodiment, spray granulation of thesolution or the suspension of the composition is achieved via two-way orthree-way nozzles.

Generally, the half maximal inhibitory concentration (IC-50 value) of acompound is a measure of the effectiveness of the compound in inhibitinga biological or a biochemical function. IC-50 value, therefore, can beconsidered a quantitative measure indicating how much of a particulardrug or any chemical substance is required to inhibit a given biologicalprocess by half (50%). Sometimes, however, GI-50 is used to symbolizethe value for the concentration that causes 50% growth inhibition. Theuse of GI-50 indicates that a correction for the cell count at time zerohas been made. An example of one formula for calculating GI-50 valuedefines GI-50 as the concentration of test compound where100×(T−T0)/(C−T0)=50, wherein T is the optical density of the test wellafter a 48 hour period of exposure to test drug is T, for example; T0 isthe optical density the test well at time zero.

The invention is illustrated in the following non-limiting examples.

VIII. EXAMPLES

The invention is illustrated in the following non-limiting examples.

Example 1 In Vitro Screening of Solid Dispersions of Compound (1-1)

Ten solid dispersions were prepared using compound (1-1) and one of fivepolymers, including hypromellose acetate succinate (HPMCAS-M),hypromellose phthalate (HPMCP-HP55), polyvinylpyrrolidone (PVP),PVP-vinyl acetate (PVP-VA), and Euragit L100-55, at both 25% and 50% ofcompound (1-1) loading, for each polymer. Solid dispersions wereprepared by a solvent evaporation method, using spray-drying followed bysecondary drying in a low-temperature convection oven. The performancewas assessed, for each solid dispersion, via a non-sink dissolutionperformance test which measured both the total amount of drug and theamount of free drug present in solution over time. Non-sink dissolutionwas chosen because it best represents the in vivo situation for lowsoluble compounds. This test included a “gastric transfer” of dispersionfrom gastric pH (0.1N NaCl, pH 1.0) to intestinal pH (FaFSSIF, pH 6.5)approximately 30 to 40 minutes after the introduction of dispersion tothe test medium, simulating in vivo conditions. [FaFSSIF is Fasted StateSimulated Intestinal Fluid, comprised of 3 mM sodium taurocholate, 0.75mM lechithin, 0.174 g NaOH pellets, 1.977 g NaH₂PO₄.H₂O, 3.093 g NaCl,and purified water qs 500 mL.] The amount of dissolved drug wasquantified using a high-performance liquid chromatography (HPLC) methodand an Agilent 1100 series HPLC. The dissolution profiles of theformulations (FIGS. 1A-1J) showed large increases in drug solubility inall dispersion candidates relative to the unformulated compound in thesame media. Of the solid dispersions, the 25% compound (1-1) in PVP, 25%compound (1-1) in HPMCAS-M, and 50% compound (1-1) in HPMCAS-Mdispersions were the most promising candidates for enhanced oralabsorption as compared to the unformulated compound, based on findinghigher levels of free drug released at intestinal pH.

Example 2 In Vivo Screening of Solid Dispersions of Compound (1-1)

The three most promising solid dispersions of compound (1-1), namely the25% compound (1-1) in PVP, 25% compound (1-1) in HPMCAS-MG, and 50%compound (1-1) in HPMCAS-M dispersions, were prepared at larger scalefor in vivo studies. Each formulation was assessed in the in vitrodissolution test described in Example 1. To ensure that thesedispersions were both amorphous and homogeneous, each dispersion wasassessed by powder x-ray diffraction (PXRD) and modulated differentialscanning calorimetry (mDSC). Additionally, to understand the effect ofwater on the glass transition temperature (Tg) for each dispersion, mDSCwas performed on samples first equilibrated at a set relative humidity(i.e., 25%, 50%, and 75% RH) for at least 18 hours. [Water can act as aplasticizer for solid dispersions and the hygroscopicity of the systemdue to the active compound or polymer can affect the amount of wateruptake by these systems.]

The non-sink dissolution results (FIGS. 2A-2C) were comparable to thosefound for the dispersions in Example 1. PXRD results (FIG. 3) showed noevidence of crystalline compound in any of the dispersions and mDSCresults (FIGS. 4A-4C) showed a single glass transition temperature (Tg)for each dispersion, indicating that each dispersion was homogeneous.The x-ray diffractomer was a Bruker D-2 Phaser. An inverse relationshipbetween Tg and relative humidity was observed for each (FIG. 5).Notably, for the 25% compound (1-1) in PVP solid dispersion equilibratedat 75% RH, there appeared to be two Tgs, indicating that phaseseparation was occurring, and this dispersion also showed a melt eventat 75% RH, suggesting that crystallization occurred during the RHequilibration (FIG. 6). This finding suggests that the 25% compound(1-1) in PVP dispersion may be less stable than the HPMCAS-Mdispersions.

To assess the bioavailability of the three dispersions, groups of malebeagle dogs (three per group) were given a 3 mg/kg dose of an aqueoussuspension of solid dispersion of compound (1-1) administered by oralgavage or a 1 mg/kg dose of compound (1-1) dissolved inwater:ethanol:polyethylene glycol (PEG) 400 (60:20:20) and administeredas an intravenous bolus into the cephalic vein. Blood samples werecollected from the jugular vein of each animal at 0 (pre-dose), 5, 15,and 30 minutes and 1, 2, 4, 8, 12, and 24 hours following intravenousadministration and at 0 (pre-dose), 15 and 30 minutes and 1, 2, 4, 8,12, and 24 hours following oral gavage administration. The amount ofcompound (1-1) present in each sample was detected using a qualifiedLC-MS/MS method with a lower limit of quantification of 0.5 ng/mL. Thearea under the plasma concentration-time curve (AUC) was determined byuse of the linear trapezoidal rule up to the last measurableconcentration without extrapolation of the terminal elimination phase toinfinity. The elimination half-life (t_(1/2)) was calculated byleast-squares regression analysis of the terminal linear part of the logconcentration-ime curve. The maximum plasma concentration (C_(max)) andthe time to C_(max) (t_(max)) were derived directly from the plasmaconcentration data. The oral bioavailability (F) was calculated bydividing the dose normalized AUC after oral administration by the dosenormalized AUC after intravenous administration and reported aspercentages (%). Results, summarized in Table 1 below, gave mean oralbioavailabilities of the 25% compound (1-1) in PVP, 25% compound (1-1)in HPMCAS-M, and 50% compound (1-1) in HPMCAS-M solid dispersions of58%, 49%, and 74%, respectively.

TABLE 1 pharmacokinetic parameters of compound (1-1) after oral (po) andintravenous (iv) administrations to dogs (the values are averages fromthree dogs) Compound (1-1) Dose & C_(max) t_(max) AUC t_(1/2) Fformulation Route (ng/L) (hr) (ng · min/mL) (hr) (%) Solution inwater:ethanol:PEG400 1 mg/kg 769 0.083 53,312 1.5 — (60:20:20) IVAqueous suspension of 25% 3 mg/kg 487 1.0 93,271 1.6 58 compound(1-1)/PVP solid PO dispersion Aqueous suspension of 25% 3 mg/kg 228 0.578,595 2.0 49 compound (1-1)/HPMCAS-M PO solid dispersion Aqueoussuspension of 50% 3 mg/kg 371 1.0 118,174 1.5 74 compound (1-1)/HPMCAS-MPO solid dispersion AUC: area under the plasma concentration-time curve;C_(max): maximum plasma concentration; F: bioavailability HPMCAS:hypromellose acetate sodium; IV: intravenous; PEG: polyethylene glycon;PO; per os, oral; PVP: polyvinylpyrrolidone; t_(max): time of C_(max);t_(1/2): plasma elimination half-life

Example 3 Preparation and Clinical Use of Capsules Containing a SolidDispersion of Compound (1-1)

A gelatin capsule of 10 mg strength was prepared for initial clinicalstudies in patients with hematologic malignancies. Based on results ofin vitro and in vivo testing of solid dispersions of compound (1-1), asdescribed in Examples 1 and 2, a 50% compound (1-1) in HPMCAS-M soliddispersion was selected for capsule development. Capsule development wasinitiated targeting a fill weight of 190 mg in a size 3 hard gelatincapsule, as this configuration would potentially allow increasing thecapsule strength by filling a larger size capsule while maintaining thepharmaceutical composition. Based on experience, four capsuleformulations were designed with different amounts of disintegrant andwith and without wetting agent. Since all four formulations showedsimilar disintegration test and dissolution test results, the simplestformulation (without wetting agent and minimum disintegrant) wasselected for manufacturing. Manufacturing process development andscale-up studies were performed to confirm the spray drying process andpost-drying times for the solid dispersion; blending parameters; rollercompaction and milling of the blend to achieve target bulk density ofapproximately 0.60 g/cc; and capsule filling conditions.

Crystalline compound (1-1) and the polymer hypromellose actate succinate(HPMCAS-M) were dissolved in acetone and spray-dried to produce soliddispersion intermediate (SDI) granules containing a 50% compound (1-1)loading. The SDI was shown by PXRD analysis to be amorphous and by mDSCanalysis to be homogeneous (i.e., single Tg under ambient conditions).The 50% compound (1-1) in HPMCAS-M solid dispersion (1000 g) andexcipients, including microcrystalline cellulose filler-binder (4428 g),croscarmellose sodium disintegrant (636 g), colloidal silicon dioxidedispersant/lubricant 156 g), magnesium stearate dispersant/lubricant(156 g), and lactose monohydrate filler (5364 g) were blended in stagesin a V-blender. The blend was them compacted and granulated to obtain abulk density of approximately 0.6 g/mL. The blend was dispensed intosize 3 hard gelatin capsules (target fill weight: 190 mg) using anautomated filling machine and finished capsules were polished using acapsule polisher machine.

Pharmacokinetic assessments were performed following oral dosing of 10mg capsules containing the 50% compound (1-1) in HPMCAS solid dispersionand results were compared with pharmacokinetic assessments performedfollowing oral dosing of administration of 4×10 mg capsules containingthe Eudragit solid dispersion of compound (1-1) to healthy volunteers

A comparison of the two pharmaceutical compositions is provided inTables 2A and 2B below. The Eudragit formulation previously wasdescribed in Example 5 in US Patent Application 2009/0012064 A1,published Jan. 8, 2009. That application noted that the Eudragit soliddispersion formulation was made by dissolving and/or dispersing thethienotriazolodiazepine of formula (A) and coating excipients, includingammonio methacrylate copolymer type B (Eudragit RS), methacrylic acidcopolymer type C (Eudragit L100-55), talc, and magnesiumaluminosilicate, in a mixture of water and ethanol. This heterogeneousmixture then was applied to microcrystalline cellulose spheres(Nonpareil 101, Freund) using a centrifugal fluidizing bed granulator toproduce granules that were dispensed into size 2 hydroxypropylmethylcellulose capsules.

In both clinical studies, blood levels of compound (1-1) were determinedusing validated LC-MS/MS methods and pharmacokinetic analyses wereperformed based on plasma concentrations of compound (1-1) measured atvarious time points over 24 hours after capsule administration. Results,summarized in Table 3 below, showed that the HPMCAS-M solid dispersionformulation had over 3-fold higher bioavailability in humans than theEudragit solid dispersion formulation based on AUCs (924*4/1140,adjusting for difference in doses administered). Additionally, based onthe observed T_(max), the HPMCAS formulation is more rapidly absorbedthan the Eudragit formulation (T_(max) of 1 h vs 4-6 h). The markedimprovement in systemic exposure with the HPMCAS-M solid dispersionformulation is unexpected.

TABLE 2A solid dispersion capsules of compound (1-1) for clinical usepharmaceutical composition containing 50% HPMCAS solid dispersion ofcompound (1-1): 10 mg strength, size 3 hard gelatin capsule CapsuleContent Ingredient Function mg Wt % Compound of formula (II) activeagent 10.0* 5.56 Hypromellose acetate carrier for solid 10.0 5.56succinate dispersion (HPMCAS-M) Lactose monohydrate filler 85.0 47.22Microcrystalline cellulose filler-binder 70.0 38.89 Croscarmellosesodium disintegrant 10.0 5.56 Collidal silicon dioxidedispersant/lubricant 2.5 1.39 Magnesium stearate dispersant/lubricantTotal 190.0 100.0

TABLE 2B pharmaceutical composition containing Eudragit L100-55 soliddispersion of compound (1-1): 10 mg strength, size 2 hard gelatincapsule Capsule Content Ingredient Function mg Wt % Compound (1-1)active agent 10.0* 3.8 Core: Microcrystalline cellulose spheres vehicle100.0 38.5 (Nonpareil 101, Freund, Inc) Compound/polymer layer: Ammoniomethacrylate copolymer, coating agent 10.8 4.2 type B (NF. PhEur)(Edragit RS, Evonik) Methacrylic acid copolymer, type C coating agent25.2 9.7 (NF)/Methacrylic acid-ethyl acrylate copolymer (1:1) type A(PhEur) (Eudragit L100-55, Evonik) Talc coating agent 88.2 33.9Magnesium aluminometasilicate coating agent 20.0 7.7 (Neuslin, FujiChemical) Triethyl citrate plasticizer 5.0 1.9 Silicon dioxidefluidizing 0.8 0.3 agent 260.0 100.0 * as anhydrate

TABLE 3 pharmacokinetic parameters following oral administration ofsolid dispersions of compound (1-1) to humans Dose Compound (1-1) # andC_(max) T_(max) AUC_(0-24h) formulation Patients Route (ng/mL) (hr) (ng· h/mL) Eudragit solid dispersion 7 40 mg 83 4 to 6 1140 formulation PO50% HMPCAS-M solid 7 10 mg 286 1 925 dispersion formulation POAUC_(0-24h): area under the OTX015 plasma concentration vs. time curveover 24 hours C_(max): maximum concentration in plasma hr: hour HPMCAS:hypromellose acetate succinate mL: milliliter ng: nanogram PO: per os,oral T_(max): time of C_(max)

Example 4 Oral Exposure in the Rat

The oral bioavailability of three formulations of solid dispersions ofcompound (1-1) was determined in rats. The three dispersions chosen werethe 25% dispersion of compound (1-1) in PVP, the 25% dispersion ofcompound (1-1) in HPMCAS-MG, and the 50% dispersion of compound (1-1) inHPMCAS-MG. The animals used in the study were Specific Pathogen Free(SPF) Hsd:Sprague Dawley rats obtained from the Central AnimalLaboratory at the University of Turku, Finland. The rats were originallypurchased from Harlan, The Netherlands. The rats were female and wereten weeks of age, and 12 rats were used in the study. The animals werehoused in polycarbonate Makrolon II cages (three animals per cage), theanimal room temperature was 21+/−3° C., the animal room relativehumidity was 55+/−15%, and the animal room lighting was artificial andwas cycled for 12 hour light and dark periods (with the dark periodbetween 18:00 and 06:00 hours). Aspen chips (Tapvei Oy, Estonia) wereused for bedding, and bedding was changed at least once per week. Foodand water was provided prior to dosing the animals but was removedduring the first two hours after dosing.

The oral dosing solutions containing the 25% dispersion of compound(1-1) in PVP, the 25% dispersion of compound (1-1) in HPMCAS-MG, and the50% dispersion of compound (1-1) in HPMCAS-MG were prepared by adding apre-calculated amount of sterile water for injection to containersholding the dispersion using appropriate quantities to obtain aconcentration of 0.75 mg/mL of compound (1-1). The oral dosing solutionswere subjected to vortex mixing for 20 seconds prior to each dose. Thedosing solution for intravenous administration contained 0.25 mg/mL ofcompound (1-1) and was prepared by dissolving 5 mg of compound (1-1) ina mixture containing 4 mL of polyethylene glycol with an averagemolecular weight of 400 Da (PEG400), 4 mL of ethanol (96% purity), and12 mL of sterile water for injection. The dosing solution containing the25% dispersion of compound (1-1) in PVP was used within 30 minutes afterthe addition of water. The dosing solutions containing the 25%dispersion of compound (1-1) in HPMCAS-MG and the 50% dispersion ofcompound (1-1) in HPMCAS-MG were used within 60 minutes of after theaddition of water. A dosing volume of 4 mL/kg was used to give doselevels of compound (1-1) of 1 mg/kg for intravenous administration and 3mg/kg for oral administration. The dosing scheme is given in Table 4.

TABLE 4 Dosing scheme for rat oral exposure study. Dose Rat Weight (mL)Test Item Route  1 236.5 0.95 Compound (1-1) intravenous  2 221 0.88Compound (1-1) intravenous  3 237.5 0.95 Compound (1-1) intravenous  4255.5 1.02 25% dispersion of compound (1-1) oral in PVP  5 224.2 0.9025% dispersion of compound (1-1) oral in PVP  6 219.2 0.88 25%dispersion of compound (1-1) oral in PVP  7 251.6 1.01 25% dispersion ofcompound (1-1) oral in HPMCAS-MG  8 240.4 0.96 25% dispersion ofcompound (1-1) oral in HPMCAS-MG  9 238 0.95 25% dispersion of compound(1-1) oral in HPMCAS-MG 10 226.6 0.91 50% dispersion of compound (1-1)oral in HPMCAS-MG 11 228.4 0.91 50% dispersion of compound (1-1) oral inHPMCAS-MG 12 228.5 0.91 50% dispersion of compound (1-1) oral inHPMCAS-MG

Blood samples of approximately 50 μL were collected into Eppendorf tubescontaining 5 μL of ethylenediaminetetraacetic acid (EDTA) solution attime points of 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after dosing,with each sample collected within a window of 5 minutes from theprescribed time point. From each sample, 20 μL of plasma was obtainedand stored at dry ice temperatures for analysis. Analysis of each samplefor the concentration of compound (1-1) was performed using a validatedliquid chromatography tandem mass spectrometry (LC-MS/MS) method with alower limit of quantitation of 0.5 ng/mL.

Pharmacokinetic parameters were calculated with the Phoenix WinNonlinsoftware package (version 6.2.1, Pharsight Corp., CA, USA) with standardnoncompartmental methods. The elimination phase half-life (t_(1/2)) wascalculated by least-squares regression analysis of the terminal linearpart of the log concentration-time curve. The area under the plasmaconcentration-time curve (AUC) was determined by use of the lineartrapezoidal rule up to the last measurable concentration and thereafterby extrapolation of the terminal elimination phase to infinity. The meanresidence time (MRT), representing the average amount of time a compoundremains in a compartment or system, was calculated by extrapolating thedrug concentration profile to infinity. The maximum plasma concentration(C_(max)) and the time to C_(max) (t_(max)) were derived directly fromthe plasma concentration data. The tentative oral bioavailability (F)was calculated by dividing the dose normalised AUC after oraladministration by the dose normalised AUC after intravenousadministration, i.e.F=(AUC(oral)/Dose(oral))/(AUC(intravenous)/Dose(intravenous))] and isreported as percentage (%).

The pharmacokinetic parameters are given in Table 5, and the plasmaconcentration versus time plots are shown in FIGS. 7 and 8.

TABLE 5 Pharmacokinetic parameters of compound (1-1) after oral andintravenous administrations. The values are an average from threeanimals. Compound Parameter 1 mg/kg 3 mg/kg oral F(%) intravenousCompound (1-1) AUC (min*ng/ml) 74698 water:ethanol:PEG C_(max) (ng/ml)730 400 (60:20:20) T_(max) (hr) 0.25 t_(1/2) (hr) 8.5 8.5 CI/F(ml/min/kg) 13.4 MRT (hr) 7.4 25% dispersion of AUC (min*ng/ml) 39920 18compound (1-1) in C_(max) (ng/ml) 77.9 PVP T_(max) (hr) 1 t_(1/2) (hr)8.5 13.8 CI/F (ml/min/kg) 75.2 MRT (hr) 18.0 25% dispersion of AUC(min*ng/ml) 35306 16 compound (1-1) in C_(max) (ng/ml) 48.3 HPMCAS-MGT_(max) (hr) 0.5 t_(1/2) (hr) 8.5 11.0 CI/F (ml/min/kg) 85.0 MRT (hr)17.1 50% dispersion of AUC (min*ng/ml) 40238 18 compound (1-1) inC_(max) (ng/ml) 67.0 HPMCAS-MG T_(max) (hr) 2 t_(1/2) (hr) 8.5 9.5 CI/F(ml/min/kg) 74.6 MRT (hr) 12.8

Example 5 Preparation of Spray Dried Dispersions

Spray dried dispersions of compound (1-1) were prepared using fiveselected polymers: HPMCAS-MG (Shin Etsu Chemical Co., Ltd.), HPMCP-HP55(Shin Etsu Chemical Co., Ltd.), PVP (ISP, a division of Ashland, Inc.),PVP-VA (BASF Corp.), and Eudragit L100-55 (Evonik Industries AG). Allspray dried solutions were prepared at 25% and 50% by weight with eachpolymer. All solutions were prepared in acetone, with the exception ofthe PVP solutions, which were prepared in ethanol. For each solution,1.0 g of solids (polymer and compound (1-1)) were prepared in 10 g ofsolvent. The solutions were spray dried using a Büchi B-290, PE-024spray dryer with a 1.5 mm nozzle and a Büchi B-295, P-002 condenser. Thespray dryer nozzle pressure was set to 80 psi, the target outlettemperature was set to 40° C., the chiller temperature was set to −20°C., the pump speed was set to 100%, and the aspirator setting was 100%.After spray drying, the solid dispersions were collected and driedovernight in a low temperature convection oven to remove residualsolvents.

Example 6 Stability with Humidity and Temperature

Acceptance T-1 month Test Procedure Criteria T = O (Initial) (storage at40° C./75% RH) Appearance AM-0002 White to off- Test Date/Ref:06Aug2012/02-41-2 Test Date/Ref: 24Sep2012/02-41- white powder 59 WhitePowder White Powder Potency AM-0028 45.0 · 55.0 wt % Test Date/Ref:25Jul2012/02-37-21 Test Date/Ref: 25Sep2012/02- (HPLC) 4HI0 50.0 49.4Individual Related AM-0029 Report results Test Date/Ref:25Jul2012/02.34-49 Test Date/Ref: 26Sep2012/02- Substances 41-64 (HPLC)RRT % Area RRT % Area No reportable related substances No reportablerelated substances Total Related AM · 0029 Report results Test Date/Ref:25Jul2012/02-34-49 Test Date/Ref: 26Sep2012/02- Substances 41-64 (HPLC)No reportable related substances No reportable related substances WaterContent AM-0030 Report results Test Date/Ref: 02Aug2012/02-41-1 TestDate/Ref: 27Sep2012/02-37- (KF) USP <921> (wt %) 99  1.52  2.53 X-RayPowder USP <941> Consistent with an Test Date/Ref: 24Jul2012/02 • 24-131Test Date/Ref: 01Oct2012/02-41- Diffraction amorphous form 73 (XRPD)Consistent with an amorphous form Consistent with an amorphous See FIG.9 form See FIG. 10 Modulated USP <891> Report individual Test Dale/Ref:24Jul2012/02-24-130 Test Date/Ref: 26Sep2012/02- Differential (n = 2 andaverage glass 37-98 Scanning replicates) transtion Replicate 1 = 134.30°C., Replicate 2 = Replicate 1 = Calorimetry temperatures (T_(g), 134.23°C., Replicate 3 = 135.28° C., 134.65° C., Replicate 2 = (mDSC) ° C.)Average = 134.60° C. 134.43° C., Average = 135.54° C. T-2 month T = 3month Test (storage at 40° C./75% RH) (storage at 40° C./75% RH)Appearance Test Date/Ref: 24Oct2012/02-37-106 TestDate/Ref:17Dec2012/02-37-119 White Powder White Powder Potency TestDate/Ref: 24Oct2012/02-37-105 Test Date/Ref: 29Nov2012/02-34-107 (HPLC)49.8 49.2 Individual Related Test Date/Ref: 24Oct2012/02-37- 105 TestDate/Ref: 29Nov2012/02-34-107 Substances RRT % Area RRT % Area (HPLC)0.68 0.06 0.68 0.07 0.77 0.06 0.77 0.09 Total Related Test Date/Ref:24Oct2012/02-37-105 Test Date/Ref: 29Nov2012/02-34-107 Substances 0.12%0.16% (HPLC) Water Content Test Date/Ref: 25Oct2012102-37-110 TestDate/Ref: 29Nov2012/02.37-116 (KF)  2.70  3.43 X-Ray Powder TestDate/Ref: 24Oct2012/02-37-107 Test Date/Ref: 17Dec2012/02-37-120Diffraction Consistent with an amorphous form Consistent with anamorphous form (XRPD) See FIG. 11 See FIG. 12 Modulated Test Date/Ref:24Oct2012/02-37-108 Test Date/Ref: 17Dec2012/02-37-121 DifferentialReplicate 1 = 135.35° C., Replicate 2 = Replicate 1 = 134.36° C.Replicate 2 = Scanning 134.93° C., Average = 135.14° C. 137.16° C.Average = 135.76° C. Calorimetry (mDSC)

Spray dried dispersions of compound (1-1) in HPMCAS-MG were assessed forstability by exposure to moisture at elevated temperature. The glasstransition temperature (Tg) as a function of relative humidity wasdetermined at 75% relative humidity, 40° C. for 1, 2 and 3 months. Thespray dried dispersion was stored in an LDPE bag inside a HDPE bottle tosimulate bulk product packaging. The data is summarized in Table 6. Attime zero, the Tg was 134° C., at 1 month the Tg was 134° C., at 2months the Tg was 135° C. and at 3 months the Tg was 134° C. and only asingle inflection point was observed for each measurement. X-raydiffraction patterns were also obtained for each sample. FIG. 9illustrates a powder X-ray diffraction profile of solid dispersions ofcompound (1-1) in HPMCAS-MG at time zero of a stability test. FIGS. 10,11 and 12 illustrate powder X-ray diffraction profiles of soliddispersions of compound (1-1) in HPMCAS-MG Spray dried dispersions ofcompound (1-1) in HPMCAS-MG were assessed for stability by exposure tomoisture at elevated temperature. The glass transition temperature (Tg)as a function of relative humidity was determined at 75% relativehumidity, 40° C. for 1, 2 and 3 months. The spray dried dispersion wasstored in an LDPE bag inside a HDPE bottle to simulate bulk productpackaging. The data is summarized in Table 6. At time zero, the Tg was134° C., at 1 month the Tg was 134° C., at 2 months the Tg was 135° C.and at 3 months the Tg was 134° C. and only a single inflection pointwas observed for each measurement. X-ray diffraction patterns were alsoobtained for each sample. FIG. 9 illustrates a powder X-ray diffractionprofile of solid dispersions of compound (1-1) in HPMCAS-MG at time zeroof a stability test. FIGS. 10, 11 and 12 illustrate powder X-raydiffraction profiles of solid dispersions of compound (1-1) in HPMCAS-MGafter 1 month, 2 months and 3 months, respectively, after exposure at40° C. and 75% relative humidity. The patterns did not show anydiffraction lines associated with compound (1-1).

Example 7 In Vitro Treatment of NSCLC Cell Lines

Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 andHOP92) were exposed to increasing doses of compound (1-1) (OncoEthix SA,Switzerland). Effect on cell viability was determined by the MTT assayafter 72 h exposure. The growth inhibition (GI) 50% values weredetermined by GraphPad Prism 5.0 software. And protein levels wereanalyzed by Western Blot using commercial antibodies. RNA was extractedwith the Qiagen RNAEasy kit and reverse-transcribed using theSuperscript First-Strand Synthesis System for RT-PCR kit followingmanufacturer's instructions. RT-PCR was performed using Fast SYBR GreenMaster Mix on a StepOnePlus Real-Time PCR System.

Compound (1-1) displayed anti-proliferative effects in bothEML4-ALK-positive NSCLC cells after 72-h exposure with GI50 values of629 and 627 nM in H2228 and H3122 cells, respectively. Interestingly,compound (1-1) was active in the EML4-ALK-negative (A49) cell line withGI50 of 432 nM. The expression of BRD4/3/2, c-MYC, BCL-2, p21 andCyclinD1 was characterized at the protein and mRNA levels in all celllines. Both compound (1-1)-sensitive and -resistant lines exhibitedsimilar basal expression levels for the aforementioned proteins.EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells,respectively. Cell signaling pathways assessment of theanti-proliferative activity showed that compound (1-1) induced atransient upregulation of STATS with a subsequent down-regulation after24 h and up to 72 h exposure, this pathway being the key downstreameffector of ALK frequently up-regulated in the rizotinibresistant celllines. Interestingly, C-MYC protein and mRNA levels appeared not alteredby 25 compound (1-1). The EML4-ALK-positive H3122 cells showeddown-regulation of N-MYC mRNA levels after compound (1-1) treatment.

These results indicate that NSCLC cell lines with genomic ALKalterations are sensitive to BET-BRD inhibition by compound (1-1), withevident anti-proliferative effects seen alongside inhibition ofdownstream signaling pathways suggesting its clinical development asanticancer agents in ALK positive NSCLC patients.

Example 8

Materials and Methods. Five established NSCLC cell lines, HOP62, HOP92,A549, H2228, and H3122 harboring different oncogenic mutations for KRAS,LKB1, TP53 and ALK (FIG. 19) were exposed to increasing doses ofcompound (1-1) (OncoEthix SA, Switzerland) for 72 h and cellproliferation was evaluated by MTT assays. Results represent themean±95% CI of at least 3 independent experiments performed intriplicate. Protein levels were analyzed by Western blot usingcommercial antibodies. For cell cycle analysis cells were stained withPI and analyzed using a FACScan flow cytometer after 24 h of treatment.RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlusReal-Time PCR System. Results represent the mean±SD of at least 2independent experiments, where *p<0.05 versus control cells (0.1% DMSO),employing Anova followed by Dunnett's Multiple Comparison Test.Concomitant compound (1-1) combination studies were performed in celllines exposed for 48 h to increasing doses of compound (1-1) alone or incombination with everolimus or crizotinib, and assessed using the Chou &Talalay method. Combination Index (CI) was determined by median effectplot analysis using CalcuSyn software. CI<1 synergy, CI=1 additivity andCI>1 antagonist effects. Results represent the median and range of 3independent experiments performed in triplicate.

In Vivo Studies:

5.10⁶ H3122 cells were injected in the flank of male athymic NMRI nudemice, and mice were randomized to 3 groups (8 mice/group) when tumorvolume reach 100 mm³; vehicle (PBS, once daily, oral continuous),Compound (1-1) (50 mg/kg/bidaily, gavage, continuous), or crizotinib (25mg/kg/daily, gavage, continuous).

Results. Compound (1-1) displays cytostatic effects in 4 out of 5 NSCLCcell lines, two of which harbor the fusion protein EML4-ALK+. In A549cells, the concurrent mutations in KRAS and LKB1 genes abrogate compound(1-1) effects. In our cell line panel, GI50 values after 72 h-exposureto compound (1-1) ranged between 0.1-0.7 μM, showing compound (1-1) isbroadly active in NSCLC cells (FIG. 13). Compound (1-1) treatment alsoresulted in a reduction in the percentage of cells in S phase (FIG. 14).In NSCLC cell lines, characterization of the expression of BRD2/3/4,C-MYC, N-MYC, BCL-2 or P21 and cyclinD1 at the protein and mRNA levelsdid not show any correlations between basal levels of these proteins andcompound (1-1) sensitivity (FIGS. 15A and 15B). That is, compound (1-1)sensitivity does not correlate with basal levels of BRDs, C-MYC, N-MYC,BCL2 or P21 in NSCLC cells. In compound (1-1)-sensitive cell lines, weobserved a rapid downregulation of C-MYC mRNA and protein levels inHOP92 cells and of N-MYC mRNA levels in HOP62 and H3122 cells after 4hours, which is maintained after 24 hours (FIGS. 16A-16C). Compound(1-1) combined with the ALK+-inhibitor crizotinib had additive effects(CI=0.9) after 48 h concomitant exposure in H2228 cells andadditive/synergistic effects with everolimus in HOP62, HOP92, A549 andH2228 cell lines (FIG. 17). In H3122 tumor bearing-mice, compound (1-1)inhibited tumor growth to a similar extent as crizotinib, an EML4-ALKinhibitor (FIG. 18). Our data indicate that SNCLC cell lines, includingthose harboring the EML4-ALK fusion gene or KRAS mutation, are sensitivein vitro and in vivo to compound (1-1) BET inhibition.

It will be appreciated by those skilled in the art that changes could bemade to the exemplary embodiments shown and described above withoutdeparting from the broad inventive concept thereof. It is understood,therefore, that this invention is not limited to the exemplaryembodiments shown and described, but it is intended to covermodifications within the spirit and scope of the present invention asdefined by the claims. For example, specific features of the exemplaryembodiments may or may not be part of the claimed invention and featuresof the disclosed embodiments may be combined. Unless specifically setforth herein, the terms “a”, “an” and “the” are not limited to oneelement but instead should be read as meaning “at least one”.

It is to be understood that at least some of the figures anddescriptions of the invention have been simplified to focus on elementsthat are relevant for a clear understanding of the invention, whileeliminating, for purposes of clarity, other elements that those ofordinary skill in the art will appreciate may also comprise a portion ofthe invention. However, because such elements are well known in the art,and because they do not necessarily facilitate a better understanding ofthe invention, a description of such elements is not provided herein.

Further, to the extent that the method does not rely on the particularorder of steps set forth herein, the particular order of the stepsshould not be construed as limitation on the claims. The claims directedto the method of the present invention should not be limited to theperformance of their steps in the order written, and one skilled in theart can readily appreciate that the steps may be varied and still remainwithin the spirit and scope of the present invention.

1. A method of treating non-small cell lung cancer in a mammalcomprising the step of: administering to a patient a pharmaceuticallyacceptable amount of a compound selected from the group consisting of:(i)(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamideor a dihydrate thereof, (ii) methyl(S)-{4-(3′-cyanobiphenyl-4-yl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate,(iii) methyl(S)-{2,3,9-trimethyl-4-(4-phenylaminophenyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate;and (iv) methyl(S)-{2,3,9-trimethyl-4-[4-(3-phenylpropionylamino)phenyl]-6H-thieno[3,2-f-][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate,or a pharmaceutically acceptable salt thereof or a hydrate or solvatethereof.
 2. (canceled)
 3. The method according to claim 1, wherein thecompound is(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate.
 4. The method according to claim 1, wherein the compound is(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamide.5. The method according to claim 3, wherein the thienotriazolodiazepinecompound is formed as a solid dispersion.
 6. The method according toclaim 5, wherein the solid dispersion comprises an amorphous(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate and a pharmaceutically acceptable salt thereof or a hydratethereof and a pharmaceutically acceptable polymer.
 7. (canceled)
 8. Themethod according to claim 5 wherein the solid dispersion exhibits anX-ray powder diffraction pattern substantially free of diffraction linesassociated with crystalline(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate.
 9. The method according to claim 6, wherein thepharmaceutically acceptable polymer is hydroxypropylmethylcelluloseacetate succinate having a(S)-2-[4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,-4]triazolo[4,3-a][1,4]diazepin-6-yl]-N-(4-hydroxyphenyl)acetamidedihydrate to hydroxypropylmethylcellulose acetate succinate (HPMCAS),weight ratio of 1:3 to 1:1.
 10. The method according to claim 5, whereinthe solid dispersion exhibits a single glass transition temperature (Tg)inflection point ranging from about 130° C. to about 140° C.
 11. Themethod according to claim 1, further comprising administering to thepatient an mTOR inhibitor.
 12. The method according to claim 11, whereinthe mTOR inhibitor is everolimus.
 13. The method according to claim 1,further comprising administering to the patient an ALK inhibitor. 14.The method according to claim 13, wherein the ALK inhibitor iscrizotinib.
 15. The method according to claim 1, wherein thenon-small-cell lung cancer is EML4-ALK positive.
 16. The methodaccording to claim 1, wherein the non-small-cell lung cancer exhibiteddown regulation of N-MYC mRNA levels after treatment.
 17. The methodaccording to claim 1, wherein the non-small-cell lung cancer expressedBRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1.
 18. The method according toclaim 1, wherein the treatment induces a transient up-regulation ofSTAT3 with a subsequent down-regulation after 24 hour and up to 72 hourexposure.
 19. The method according to claim 1, wherein thenon-small-cell lung cancer is EML4-ALk negative.
 20. The methodaccording to claim 19, wherein the non-small-cell lung cancer expressedBRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1.
 21. The method according toclaim 19, wherein the treatment induces a transient up-regulation ofSTAT3 with a subsequent down-regulation after 24 hour and up to 72 hourexposure.
 22. The method according to claim 1, wherein thenon-small-cell lung cancer has a mutation in the KRAS gene.
 23. Themethod according to claim 1, wherein the non-small-cell lung cancer hasa mutation in the LKB1 gene.
 24. The method according to claim 1,wherein NMYC is downregulated.
 25. The method according to claim 1,wherein HEXIM is upregulated.